European Journal of Pharmacology 545 (2006) 73–80www.elsevier.com/locate/ejphar

Review

Predicting Alzheimer dementia in mild cognitive impairment patientsAre biomarkers useful?

Barbara Borroni a,⁎, Monica Di Luca b, Alessandro Padovani a,c

a Department of Medical Sciences, University of Brescia, Italyb Centre of Excellence for Neurdegenerative Disorders, Department of Pharmacological Sciences, University of Milan, Italy

c Centre for Behavioural Disturbances and Neurodegenerative Disorders, EULO, Italy

Accepted 13 June 2006Available online 16 June 2006

Abstract

A correct clinical diagnosis in the early stage of Alzheimer disease is not only of importance given the current available treatment withacetylcholine esterase inhibitors, but would be the basis for disease-modifying therapy slowing down or arresting the degenerative process.Moreover, in the last years, several efforts have been made to determine if a patient with mild cognitive impairment has incipient Alzheimerdisease, i.e. will progress to Alzheimer disease with dementia, or have a benign form of mild cognitive impairment. In this review, the recentpublished reports regarding progress in early and preclinical Alzheimer disease diagnosis are discussed and the role of peripheral andcerebrospinal fluid biomarkers highlighted. Approaches combining panels of different biomarkers show promise for discovering profiles that arecharacteristic of Alzheimer disease, even in the pre-symptomatic stage. More work is needed but available novel perspectives offered by recentintroduced technologies shed some lights in identifying incipient Alzheimer disease in mild cognitive impairment subjects.© 2006 Elsevier B.V. All rights reserved.

Keywords: Alzheimer disease; Mild cognitive impairment; Biomarker; Cerebrospinal fluid; Amyloid precursor protein; Platelet

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 742. Diagnostic issues in mild cognitive impairment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 743. Biological markers and early diagnosis of Alzheimer disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 744. Peripheral biormarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

4.1. Abeta peptide in plasma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 754.2. Amyloid precursor protein forms in platelets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 754.3. Amyloid cascade in platelets as a combination of biomarkers for early Alzheimer disease diagnosis . . . . . . . . . . . . . 76

5. CSF biomarkers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 765.1. Abeta levels in CSF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 765.2. Total tau and phospho-tau levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 765.3. Combination of CSF Abeta 42 and CSF tau levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 775.4. Oxidative stress biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

6. Conclusive remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

⁎ Corresponding author. Department of Neurology, University of Brescia, Pza Spedali Civili, 1, 25100 Brescia, Italy. Tel.: +39 0303995632.E-mail address: bborroni@inwind.it (B. Borroni).

0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved.doi:10.1016/j.ejphar.2006.06.023

74 B. Borroni et al. / European Journal of Pharmacology 545 (2006) 73–80

1. Introduction

Alzheimer disease represents the most common neurode-generative disease worldwide accounting for 60% to 70% ofcases of progressive cognitive impairment in elderly patients.The prevalence of dementia of the Alzheimer type doublesevery 5 years after the age of 60 increasing from a prevalence of1% among those 60- to 64-year-old up to 40% of those aged85 years and older (Von Strauss et al., 1999; Di Carlo et al.,2002). The characteristic findings at the microscopic level aredegeneration of the neurons and their synapses together withextensive amounts of senile plaques and neurofibrillary tangles(Ghiso and Frangione, 2002). The degenerative process prob-ably starts 20–30 years before the clinical onset of Alzheimerdisease. During this preclinical period, the number of plaquesand tangles increases, and at a certain threshold the first symp-toms, most often impairment of episodic memory, appear.

The introduction of acetylcholine esterase inhibitors assymptomatic treatment has highlighted the importance of diag-nostic markers for Alzheimer disease (Cummings and Cole,2002; DeKosky and Marek, 2000). Such compounds willprobably be most effective in the earlier stages of the disease,before neurodegeneration is too severe and widespread. Theincreased general knowledge on Alzheimer disease in the po-pulation, and the awareness of the possibilities for drug treat-ment, has also made patients seek medical advice early in thedisease, when the characteristic clinical picture of dementia israther subtle in the early stage and barely noticeable.

There is no clinical method to accurately identify Alzheimerdisease in the very early phase and to determine which at riskcases will progress to Alzheimer disease except for a very longclinical follow-up period (Frisoni et al., 2004; Nestor et al., 2004).According to current diagnostic criteria, in fact, Alzheimer dis-ease cannot be diagnosed clinically before the disease has pro-gressed so far that dementia is present. This means that thesymptoms must be severe enough to significantly interfere withwork and social activities or relations, but the observationsreported above highlight the great clinical need for diagnosticinstruments to identify early Alzheimer disease.

2. Diagnostic issues in mild cognitive impairment

Available evidence suggests that mild dementia is rarelydiagnosed and even moderately severe dementia is under-recognized in clinical practice (Callahan et al., 1996). Thus,diagnostic accuracy is much lower at the earlier clinical, andespecially pre-symptomatic, stages of the disease. In recentyears, the clinical phase of Alzheimer disease with mild me-mory impairment but without overt dementia, the so-calledpreclinical stage, has gained increased attention in the medicalcommunity as it truly represents a high-risk pre-dementia state(Nestor et al., 2004; Frisoni et al., 2003). Among other terms,mild cognitive impairment has been considered to better re-present the clinical manifestation of incipient Alzheimer diseasethan other, over-inclusive terms (Petersen et al., 2001).Clinically, mild cognitive impairment is defined as an impair-ment in one or more cognitive domains (typically memory), or

an overall mild cognitive decline that is greater than would beexpected for an individual's age or education, but that is in-sufficient to interfere with social and occupational functioning.Patients fulfilling these criteria have a 5- to 10-fold greater riskof developing clinical Alzheimer disease within 3 to 5 years(Luis et al., 2003). Further, it has been suggested that mildcognitive impairment concept captures a high proportion ofpatients with Alzheimer disease at the prodromal stage asconfirmed by pathological evidence (Morris et al., 2001;Mufson et al., 1999). Several observations, however, indicatethat not all mild cognitive impairment patients deteriorate overtime, thus suggesting that mild cognitive impairment is rather aheterogeneous condition (Chetelat and Baron, 2003; Fisk et al.,2003). A report from an international group of experts on mildcognitive impairment emphasized that non-amnestic presenta-tions of mild cognitive impairment exist and might progress todementing illnesses other than Alzheimer disease (Petersenet al., 1999). Moreover, some mild cognitive impairment pa-tients do not have prodromal dementia of any type but insteadhave reversible impairment; overtime, these individuals mayrevert to normal whereas others may have a stable impairment(Larrieu et al., 2002). Pathological and clinical data have ac-cumulated in the last few years showing that some biologicalindicators of Alzheimer disease might be used to distinguishthose mild cognitive impairment patients who will progressfrom those who will not.

Thus, new diagnostic tools for the diagnosis of early andpreclinical Alzheimer disease would be of great importance. Ourgoal with this review concerns early recognition of Alzheimerdisease focusing on the use of biological markers in identifying,among individuals at risk those who will progress to Alzheimerdisease (Petersen et al., 1999).

3. Biologicalmarkers and early diagnosis ofAlzheimerdisease

Tremendous efforts have been made in recent years toidentify the neuropathological, biochemical, and genetic bio-markers of diseases so that the diagnosis could be established inearlier stages.

The search of relevant biomarkers of Alzheimer disease inliving patients has been an active part of clinical research for thelast decades. The assumption for such enterprise is that a bio-marker provides at least an indirect link to the disease process orideally directly relates to the primary mechanism of the disease.According to a recent consensus report (Growdon et al., 1998),an ideal biomarker for Alzheimer disease should fulfil thefollowing criteria: a) detect a fundamental feature of the neuro-pathology; b) be validated in neuropathologically confirmedcases; c) have a sensitivity higher than 85% for detectingAlzheimer disease and have a specificity of more than 75% fordistinguishing Alzheimer disease from other causes of demen-tia, preferably established by two independent studies appro-priately powered; d) be precise, reliable and inexpensive; e) beconvenient to use and not harmful to the patient. A well-cha-racterized biological marker that fulfils these requirementswould have several advantages. An ideal biological markerwould identify Alzheimer disease cases at a very early stage of

75B. Borroni et al. / European Journal of Pharmacology 545 (2006) 73–80

the disease, before the cognitive symptoms are found in neu-ropsychological tests, and before there is a clear-cut degener-ation in brain imaging studies. Indeed, antecedent markerscould identify those individuals with Alzheimer disease inthe pre-dementia stage or at risk of developing symptomaticAlzheimer disease and might predict those patients who mightbenefit most from the development of disease-modifying orpreventive interventions. With regard to Alzheimer disease,ante-mortem biochemical markers have been sought for years indifferent peripheral tissues and cells such as erythrocytes (Bosmanet al., 1991), lymphocytes (Pirttila et al., 1992), urine (Linderet al., 1993; Ghanbari and Ghanbari, 1998), hair (De Berker et al.,1997; Bonafe et al., 1998), and skin (Soininen et al., 1992;Heinonen et al., 1994). In fact, despite the attempts to identifybiochemical markers related to Alzheimer disease which have ledto promising results, the field is far from being satisfied, sincethe diagnostic value of the most hitherto proposed biochemicalmarkers has been limited by considerable overlap betweenAlzheimer disease patients, patients with other dementing ill-nesses, and normal subjects.

In the last decade, along with a better definition of the mainpathogenetic characteristics of Alzheimer disease, there havebeen consistent findings with regard to selected “peripheral”and cerebrospinal fluid (CSF) biomarkers, related to the “keyplayers” of Alzheimer disease pathology (i.e. degeneration ofneurons and synapses, disturbance in the Amyloid PrecursorProtein (APP) metabolism and its subsequent deposition insenile plaques, and the hyperphosphorylation of tau with sub-sequent formation of neurofibrillary tangles),which have providedrobust accuracy values reaching high specificity and sensitivitylevels, even in early or preclinicalAlzheimer disease, and opened anew era in the diagnostic approach to Alzheimer disease. Thefollowing sections will review the main findings on these bio-markers focusing on their diagnostic and predictive value.

4. Peripheral biormarkers

4.1. Abeta peptide in plasma

In the last decade, several attempts have been performed toidentify peripheral markers by using plasma, serum or circulatingcells. In particular, because amyloid plaques are a defining featureof Alzheimer disease neuropathology, and Abeta can be detectedplasma, its measure is a compelling candidate biomarker forAlzheimer disease. Abeta occurs in two prominent forms, con-taining 40 (Abeta 40) or 42 (Abeta 42) amino acids depending onthe C terminus.

Numerous investigators have tried to assess plasma Abeta 40and Abeta 42 levels, and up to date, several cross-sectionalstudies and two longitudinal studies investigated plasma Abetameasures in Alzheimer disease patients and controls. Moststudies but one have shown that plasma Abeta 40 and Abeta 42levels are not different in Alzheimer disease and control groups,thus minimising its diagnostic usefulness (Mehta et al., 2000;Tamaoka et al., 1996; Vanderstichele et al., 2000; Fukumotoet al., 2003). However, recent longitudinal studies showed thathigh plasma Abeta 42 levels were a risk factor for developing

Alzheimer disease (Mayeux et al., 1999). Similarly, patientswith Alzheimer disease or individuals who developed Alzhei-mer disease within 5 years after plasma collection were found tohave higher levels of plasma Abeta 42 levels than individualwho did not become demented while a significant decline over3 years follow-up was observed in Alzheimer disease patients(Mayeux et al., 2003). In sum, the findings on plasma Abeta 42levels indicate that this biomarker is not sensitive and specificfor the early diagnosis, though it might be used in selected casesfor predicting Alzheimer disease risk.

4.2. Amyloid precursor protein forms in platelets

Among the different peripheral cells expressing amyloidprecursor protein (APP) forms, platelets are particularly in-teresting since they show concentrations of its forms equivalentto those found in the brain. Several hypotheses have been putforward on the possible physiological role of APP in the blood:it is now known that soluble APP containing the KPI domain ishighly homologous to protease Nexin II, and it inhibits theactivity of the blood coagulation factors (Smith et al., 1990).The appropriateness to use platelets, as a cell mirroring someneurochemical processes, finds its rationale in the numeroussimilar features of platelets and neurones: platelets store andrelease neurotransmitters, express appropriate neurotransmittertransporters and some neurone-related proteins. On this line,different authors reported abnormalities in the platelets' phy-siology and function in Alzheimer disease (Zubenko et al.,1987; Davies et al., 1988; Blass and Gibson, 1992; Matsushimaet al., 1995; Ferrarese et al., 2000; Zoia et al., 2004). Moreover,a number of laboratories independently described alterations inAPP metabolism in the platelets of Alzheimer disease patientswhen compared to control subjects matched for demographiccharacteristics (Di Luca et al., 1996; Davies et al., 1997;Rosenberg et al., 1997). In particular, two research groups havereported that Alzheimer disease is associated with a decrease inthe amount of the higher (130 kDa) band compared to the lower(110 kDa band) (Baskin et al., 2000; Padovani et al., 2001a,b).

Reduction in theAPP form ratio (130 kDa/110 kDa forms)wasfound to be correlated with disease severity and progression(Borroni et al., 2002; Padovani et al., 2001a,b). Sensitivity andspecificity for Alzheimer disease diagnosis was in the 80–95%range, based on post-hoc cutoff scores (Padovani et al., 2001a,b).Thus, the APP ratio has the potential to be of clinical usefulnessto improve diagnostic accuracy or guide disease-modifyingtherapy. It fulfils most of the criteria recently settled by a con-sensus conference (The Ronald and Nancy Reagan ResearchInstitute of the Alzheimer's Association and the National Instituteon AgingWorking Group, 1998) regarding diagnostic biomarkersfor Alzheimer disease. In fact, the APP ratio reflects a neuro-pathologic feature of Alzheimer disease, is able to detect Alzhei-mer disease early in its course, allows to differentiate Alzheimerdisease from other types of dementia, is reliable and non-invasive,and has a sensitivity and specificity higher than 80%.

It has recently been observed that platelet APP ratio is alsoable to predict the conversion to dementia of the Alzheimertype in patients with the so-called mild cognitive impairment. In

76 B. Borroni et al. / European Journal of Pharmacology 545 (2006) 73–80

fact, sensitivity and specificity for prediction of conversion toAlzheimer disease at a 2-year follow-up was 83% and 71%respectively (Padovani et al., 2002; Borroni et al., 2003).

4.3. Amyloid cascade in platelets as a combination ofbiomarkers for early Alzheimer disease diagnosis

In platelets it is possible to measure the levels of the threemolecular key-elements of the amyloid cascade, namely APPforms as well as beta-secretase (beta-site-APP cleaving enzyme,BACE) enzyme, responsible for amyloidogenic pathway, andalpha-secretase (A Disintegrin And Metalloprotease, ADAM10)responsible for non-amyloidogenic metabolism (Colciaghi et al.,2002, 2004).

Indeed, in the platelets of mild Alzheimer disease patients wewere able to show an alteration of the specific APP forms pa-ralleled by a decreased expression level and activity of ADAM10as well as an increased BACE activity, when compared to controlsubjects (Colciaghi et al., 2004).

In a recent study, we applied artificial neural networks on thisset of biomarkers for Alzheimer disease, designed on the amyloidcascade, and this approach improved by more than 10% theaccuracy of the early diagnosis of the disease, being the overallaccuracy higher than 97% in blind testing (Di Luca et al., 2005).Thus, the simultaneous use of different biomarkers, each of themexploring a component of a complex pathophysiological pathway,has the intrinsic advantage to capture more information linked tothe disease under study compared to a single biomarker approach.The concomitant use of different factors may decrease the risk ofrandom variation of a single factor obscuring the true signal.

Studies performed on a large sample with pre-symptomaticsubjects or patients with other kind of dementia, will be usefulto confirm the diagnostic value of this approach.

5. CSF biomarkers

The CSF is in direct contact with the extracellular space ofthe brain, and thus biochemical changes in the brain are reflectedin the CSF (Blennow, 2004), thus candidate biomarkers forAlzheimer disease should be a protein, or molecule, reflect thecentral pathogenic processes in the brain, i.e. the neuronal de-generation, the aggregation of beta-amyloid with subsequentdeposition in plaques, and the hyperphosphorylation of tau withsubsequent formation of tangles. Hitherto, three CSF biomarkers,total-tau, Abeta-isoforms, in particular the 42 amino acid variant,and different phospho-tau epitopes, have been found to have thehighest diagnostic potential.

5.1. Abeta levels in CSF

Abeta peptide is the main protein constituent of plaques.Abeta is constitutively generated by proteolytic cleavage of itsprecursor, the APP (Masters et al., 1985).

APP, as reported above, is differently cleaved in Alzheimerdisease patients compared to healthy controls, leading to an in-crease of the terminal forms of Abeta, i.e. Abeta 40 and Abeta 42.Abeta 42 peptide aggregates more rapidly and deposits in diffuse

and senile plaques (Iwatsubo et al., 1994; Tamaoka et al., 1994).These data made it logical to develop ELISAmethods specific forAbeta peptides dosage (Motter et al., 1995; Vanderstichele et al.,2005).

Initial studies focusing on Abeta 40 failed to evidencesignificant difference between Alzheimer disease and normals(Sjogren et al., 2003; Palmert et al., 1990; Van Nostrand et al.,1992). In spite of a slight decrease, there was large overlapbetween Alzheimer disease patients and controls. At variance,majority of the studies showed that Abeta 42 concentrations aredecreased in CSF of Alzheimer disease patients, to about 50%of control levels (Andreasen et al., 2001; Blennow and Hampel,2003). The sensitivity of Abeta 42 reported in most reportsvaries from 55 to 100% (mean 86%) whereas specificity todistinguish patients with Alzheimer disease from controls variesfrom 67 to 100% (mean 89%). Accuracy data between Alzhei-mer disease and other dementias are relatively limited as de-creased CSFAbeta 42 levels are found in patients with VascularDementia, Fronto-Temporal Dementia, Creutzfeld–Jakob Dis-ease, Amiotrophic Lateral Sclerosis, and Multiple SystemicAtrophy, thus suggesting a limited value in the differentialdiagnosis of dementia. Reduced CSF levels of Abeta 42 havealso been found in early Alzheimer disease (Riemenschneideret al., 2000) and in cases with mild cognitive impairment(Andreasen et al., 1999), suggesting that a decrease of Abeta 42levels might be of help in identifying the early stage of Alzhei-mer disease. A few studies have evaluated the performance ofAbeta 42 in mild cognitive impairment cases focusing on themagnitude of the index test to predict conversion to Alzheimerdisease within a given interval; most studies but one haveshown that there is a difference among mild cognitive impair-ment patients between those who convert to Alzheimer diseaseand those who do not and that low levels of Abeta 42 signi-ficantly predict conversion to Alzheimer disease in mild cog-nitive impairment patients with high sensitivity and specificity(Maruyama et al., 2001; Hampel et al., 2004a,b; Skoog et al.,2003). These findings support the view that disturbance of themetabolism of Abeta 42 is present very early in the diseaseprocess of Alzheimer disease, and that the measurement of CSFlevels of Abeta 42 holds the premises to be a reliable andpredictive diagnostic test.

Moreover, studies using mass spectrometry have found thatthere is a heterogeneous set of Abeta peptides in CSF and it ispossible to separate several C-terminally truncated Abeta peptides,including Abeta 1-37, Abeta 1-38, Abeta 1-39, Abeta 1-40 andAbeta 1-42 in human CSF. Similar data has been found usingsurface-enhanced laser desorption/ionization time-of-flight massspectrometry (Lewczuk et al., 2004). Increased CSF levels of bothAbeta 1-40 and Abeta 1-38, together with a decrease in Abeta 1-42were found in Alzheimer disease (Wiltfang et al., 2002). Furtherstudies needed to examine the diagnostic potential of these Abetaspecies will be performed in prodromal Alzheimer disease stages.

5.2. Total tau and phospho-tau levels

One of the major neuropathological hallmarks of Alzheimerdisease is neurofibrillary tangles which are composed of paired

77B. Borroni et al. / European Journal of Pharmacology 545 (2006) 73–80

helical filaments, the principal protein subunit of paired helicalfilaments being abnormally hyperphosphorylated tau (p-tau).Total tau (t-tau) and truncated forms of monomeric and phos-phorylated tau are released and can be found in the CSF.

The t-tau, a general marker of neuronal destruction, has beenintensely studied in more than 2000 Alzheimer disease patientsand 1000 age-matched elderly controls over the last 10 years.The most consistent finding is a significant increase of CSFlevels of t-tau protein in Alzheimer disease (about 300% com-pared to normal controls) (Sjogren et al., 2001). Specificitylevels were between 65 and 86% and sensitivity between 40 and86%. In mild dementia, the potential of CSF t-tau protein todiscriminate between Alzheimer disease and normal aging isalso high, with a mean sensitivity of 75% and specificity of85%. The usefulness of t-tau is, however, limited by the rela-tively low power in differentiating Alzheimer disease fromother dementing illnesses (Sunderland et al., 2003; Hulstaertet al., 1999).

Nevertheless, there is increasing evidence that t-tau might beof help for the prediction of AD in mild cognitive impairmentsubjects. In fact, t-tau not only is increased in patients sufferingfrom mild cognitive impairment but particularly in those whoconverted to Alzheimer disease during follow-up (Maruyamaet al., 2001; Hampel et al., 2004a,b) and different studies havereported that t-tau predicts conversion to Alzheimer diseasewith a high sensitivity and specificity (Andreasen et al., 1999;Hampel et al., 2004a,b). More recently, immunoassays tospecifically detect phosphorylated tau (p-tau) at different epi-topes (threonine 231, serine 199, serine 396, serine 404, thre-onine 181) have been developed. Evidence from recent studiesindicates that all these biomarkers with minimal variation fordifferent p-tau epitopes, are more sensitive and specificallyrelated to Alzheimer disease. In fact, CSF p-tau231 distin-guished between Alzheimer disease patients and subjects withother neurological disorders with a sensitivity of 85% and aspecificity of 97% (Itoh et al., 2001, Buerger et al., 2002).

Similar discriminative values have been obtained by usingeither p-tau199 or p-tau181 (Hampel et al., 2004a,b). To date, afew studies have investigated p-tau inmild cognitive impairmentsubjects reporting increased levels of p-tau in mild cognitiveimpairment subjects and a significant correlation between base-line levels and subsequent cognitive decline (Blennow andHampel, 2003).

5.3. Combination of CSF Abeta 42 and CSF tau levels

Since diagnostic accuracy of CSF tau and Abeta 42 aloneis not clear-cut in the differentiation of Alzheimer diseasefrom other clinically relevant dementing disorders, a combi-nation of both markers has been proposed to increase ac-curacy power. The potential of the combination of these CSFbiomarkers has been evaluated in several studies comparingAlzheimer disease with normal controls and other dementedgroups such as Fronto-Temporal Dementia, Lewy Body De-mentia, Vascular Dementia (Andreasen et al., 2001; Hulstaertet al., 1999; Galasko et al., 1997). These studies have re-ported slightly higher sensitivity and specificity values (85–

89% and 80–90% respectively) for the combination than fort-tau and Abeta 42 alone in Alzheimer disease patients versusnormal controls. However, other studies, contrasting Alzhei-mer disease with other dementia, showed that the combinedassays yielded rather low discriminative power indicating thatthe combination may not sufficiently improve differential di-agnosis between Alzheimer disease and other clinically relevantdementias.

On the contrary, a recent study, comparing patients withearly-onset Alzheimer disease, normal controls and patientswith Fronto-Temporal Dementia, by measuring CSF Abeta 42,total tau and p-tau181, showed that the combination of Abeta 42and p-tau181 allowed Alzheimer disease patients to be distin-guished from Fronto-Temporal Dementia patients with a sen-sitivity of 72% and a specificity of 93%, thus supporting the claimthat this specific combination might eventually be helpful inclinical practice in early Alzheimer disease diagnosis (Schoon-enboom et al., 2004, Maddalena et al., 2003).

5.4. Oxidative stress biomarkers

Increased markers of protein, lipid, and nucleic acid ox-idation and reduced activities of antioxidant enzymes inAlzheimer disease brain support a role for oxidative stress inthe neurodegeneration, as well as their usefulness as candidatebiomarkers in Alzheimer disease (Bonilla et al., 1999).

Indeed, in addition to the presence of senile plaques andneurofibrillary tangles, the Alzheimer disease brain exhibitsevidence for oxygen radical-mediated damage, a situation com-monly known as oxidative stress. However, the ability to di-rectly implicate this mechanism in Alzheimer disease has been adifficult task for several reasons. First, most of the analyticalapproaches used to investigate oxidative stress turned out to beunreliable. Second, the majority of the published studies havebeen performed in post-mortem tissues with advanced disease,leaving open the question as to whether oxidative stress is anearly event or a common final step secondary to the degene-rative process (Pratico et al., 2000).

The accumulated information points toward an earlierinvolvement than previously thought of oxidative stress in thepathogenesis of the disease, making this a potential target forearly diagnosis, especially in subjects at high risk for deve-loping Alzheimer disease.

Free radical damage of proteins and polyunsaturated fattyacids results in modified forms that can be measured in fluids asmarkers of oxidation state.

In particular, F2-isoprostanes, one group of lipid peroxida-tion products derived from arachidonic acid, were elevated inAlzheimer disease brain and CSF, with little overlap withcontrol subjects and have been demonstrated useful as in vivobiomarkers (Pratico et al., 1998; Montine et al., 1999; Praticoand Delanty, 2000).

There is a broad agreement that increased CSF levels ofF2-isoprostanes also are present in early Alzheimer disease,demonstrating their potential aid in the assessment of antioxidantexperimental therapeutics and laboratory diagnosis in preclinicalAlzheimer disease.

78 B. Borroni et al. / European Journal of Pharmacology 545 (2006) 73–80

6. Conclusive remarks

In the last few years impressive advances in many areas ofbasic and clinical neurosciences are witnessed, but few fieldsexperienced greater progress than those dealing with Alzheimerdisease. A wide range of biological markers with possible re-levance to early AD diagnosis have been developed in the lastdecades, though in most cases the results have been dis-appointing and still inconclusive. On the other hand, there is avast literature on selected biomarkers more closely related withthe main pathogenetic mechanisms of Alzheimer disease whichindicate that either peripheral markers or CSF or such as plateletAPP isoform ratio, CSF Abeta 42, CSF protein tau, or CSFphospho-tau, or markers of oxidative stress might be of great aidin clinical practice, though it has to be acknowledged that nosingle laboratory diagnostic test yet identified permits accurateand reliable ante-mortem diagnosis comparable to autopsy studieswhich remain the gold standard for confirming the diagnosis.

Up to now, some studies have evaluated the diagnostic valueof biomarkers to identify mild cognitive impairment cases thatlater will progress to Alzheimer disease with dementia, i.e. have‘preclinical’ or incipient Alzheimer disease. A pitfall with thesestudies is that since only around 15% of mild cognitive impair-ment cases progress to Alzheimer disease each year, a veryextensive follow-up period (>5 years) would be needed to becertain that mild cognitive impairment patients will not developdementia, i.e. have stable mild cognitive impairment.

Therefore, like in other areas of medicine, diagnostic bio-markers should not be used as isolated tests. Given the multi-plicity of pathophysiological processes implicated in Alzheimerdisease, the diagnostic accuracy of biomarkers may be im-proved by combining several serum or plasma markers, therebycreating a more robust biomarker profile characteristic of Alzhei-mer disease. Indeed, it might be well true that a single neuro-chemical marker unique to Alzheimer disease would never beidentified, and that an acceptable diagnostic accuracy would bereached only through the combination of different biomarkerstapping different pathogenetic steps.

Furthermore, new mass-spectrometric techniques suchas matrix-assisted laser-desorption-ionisation time-of-flight(MALDI-ToF) mass spectrometry (MS) and electrospray ion-isation (ESI) have been developed and already been used fordetecting biomarker profiles in early Alzheimer disease. Thesetechniques will be performed for predicting Alzheimer disease inmild cognitive impairment subjects.

Although more studies are needed to determine the diag-nostic performance of biomarkers to identify incipient and earlyAlzheimer disease, we suggest that these markers have a clinicalpotential help to resolve this diagnostic challenge. Early diag-nosis of Alzheimer disease is not only of importance to be ableto initiate symptomatic treatment with acetylcholine esteraseinhibitors, but will be the basis for initiation of treatment withdrugs aimed at slowing down or arresting the degenerativeprocess, such as secretase inhibitors, if these prove to affectAlzheimer disease pathology and to have a clinical effect.

Future studies will confirm whether the challenge of earlydiagnosis of Alzheimer disease is not a chimera, but the prom-

ising perspective of the development of predictive markers ofAlzheimer disease, in addition to emerging therapeutic strat-egies, seems more concrete than ever.

References

Andreasen, N., Hesse, C., Davidsson, P., Minthon, L., Wallin, A., Winblad,B., Vanderstichele, H., Vanmechelen, E., Blennow, K., 1999. Cerebrospi-nal fluid beta-amyloid(1-42) in Alzheimer disease: differences betweenearly- and late-onset Alzheimer disease and stability during the course ofdisease. Arch. Neurol. 56, 673–680.

Andreasen, N., Minthon, L., Davidsson, P., Vanmechelen, E., Vanderstichele, H.,Winblad, B., Blennow, K., 2001. Evaluation of CSF-tau and CSFAbeta42 asdiagnostic markers for Alzheimer disease in clinical practice. Arch. Neurol. 58,373–379.

Baskin, F., Rosenberg, R.N., Iyer, L., Hynan, L., Cullum, C.M., 2000. PlateletAPP isoform ratios correlate with declining cognition in AD. Neurology 54,1907–1909.

Blass, J.P., Gibson, G.E., 1992. Nonneural markers in Alzheimer disease.Alzheimer Dis. Assoc. Disord. 6, 205–224.

Blennow, K., 2004. CSF biomarkers for mild cognitive impairment. J. Intern.Med. 256, 224–234.

Blennow, K., Hampel, H., 2003. CSF markers for incipient Alzheimer's disease.Lancet. Neurol. 2, 605–613.

Bonafe, J.L., Cambon, L., Ousset, P.J., Pech, J.H., Bellet, H., Vallat, C., 1998.Abnormal hair samples from patients with Alzheimer disease. Arch.Dermatol. 134, 1300.

Bonilla, E., Tanji, K., Hirano, M., Vu, T.H., DiMauro, S., Schon, E.A., 1999.Mitochondrial involvement in Alzheimer's disease. Biochim. Biophys.Acta. 1410, 171–182.

Borroni, B., Colciaghi, F., Corsini, P., Akkawi, N., Rozzini, L., Del Zotto, E.,Talarico, G., Cattabeni, F., Lenzi, G.L., Di Luca, M., Padovani, A., 2002.Early stages of probable Alzheimer disease are associated with changes inplatelet amyloid precursor protein forms. Neurol. Sci. 23, 207.

Borroni, B., Colciaghi, F., Caltagirone, C., Rozzini, L., Broglio, L., Cattabeni,F., Di Luca, M., Padovani, A., 2003. Platelet amyloid precursor proteinabnormalities in mild cognitive impairment predict conversion to dementiaof Alzheimer type: a 2-year follow-up study. Arch. Neurol. 60, 1740–1744.

Bosman, G.J., Bartholomeus, I.G., De Man, A.J., Van Kalmthout, P.J., De Grip,W.J., 1991. Erythrocyte membrane characteristics indicate abnormal cellularaging in patients with Alzheimer's disease. Neurobiol. Aging. 12, 13–18.

Buerger, K., Teipel, S.J., Zinkowski, R., Blennow, K., Arai, H., Engel, R.,Hofmann-Kiefer, K., McCulloch, C., Ptok, U., Heun, R., Andreasen, N.,DeBernardis, J., Kerkman, D., Moeller, H., Davies, P., Hampel, H., 2002.CSF tau protein phosphorylated at threonine 231 correlates with cognitivedecline in mild cognitive impairment subjects. Neurology 59, 627–629.

Callahan, C.M., Hall, K.S., Hui, S.L., Musick, B.S., Unverzagt, F.W., Hendrie, H.C.,1996. Relationship of age, education, and occupation with dementia among acommunity-based sample of African Americans. Arch. Neurol. 53, 134–140.

Chetelat, G., Baron, J.C., 2003. Early diagnosis of Alzheimer's disease:contribution of structural neuroimaging. Neuroimage 18, 525–541.

Colciaghi, F., Borroni, B., Pastorino, L., Marcello, E., Zimmermann, M.,Cattabeni, F., Padovani, A., Di Luca, M., 2002. [alpha]-Secretase ADAM10as well as [alpha]APPs is reduced in platelets and CSF of Alzheimer diseasepatients. Mol. Med. 8, 67–74.

Colciaghi, F., Marcello, E., Borroni, B., Zimmermann, M., Caltagirone, C.,Cattabeni, F., Padovani, A., Di Luca, M., 2004. Platelet APP, ADAM 10 andBACE alterations in the early stages of Alzheimer disease. Neurology 62,498–501.

Cummings, J.L., Cole, G., 2002. Alzheimer disease. JAMA 287, 2335–2338.Davies, T.A., Drotts, D., Weil, G.J., Simons, E.R., 1988. Flow cytometric mea-

surements of cytoplasmic calcium changes in human platelets. Cytometry 9,138–142.

Davies, T.A., Long, H.J., Sgro, K., Rathbun, W.H., McMenamin, M.E., Seetoo, K.,Tibbles, H., Billingslea, A.M., Fine, R.E., Fishman, J.B., Levesque, C.A., Smith,S.J.,Wells, J.M., Simons, E.R., 1997.ActivatedAlzheimer disease platelets retainmore beta amyloid precursor protein. Neurobiol. Aging. 18, 147–153.

79B. Borroni et al. / European Journal of Pharmacology 545 (2006) 73–80

De Berker, D., Jones, R., Mann, J., Harwood, G., 1997. Hair abnormalities inAlzheimer's disease. Lancet 350, 34.

DeKosky, S.T., Marek, K., 2000. Looking backward to move forward: earlydetection of neurodegenerative disorders. Science 302, 830–834.

Di Carlo, A., Baldereschi, M., Amaducci, L., Lepore, V., Bracco, L., Maggi, S.,Bonaiuto, S., Perissinotto, E., Scarlato, G., Farchi, G., Inzitari, D., ILSAWorking Group, 2002. Incidence of dementia, Alzheimer's disease, andvascular dementia in Italy. The ILSA Study. J. Am. Geriatr. Soc. 50, 41–48.

Di Luca, M., Grossi, E., Borroni, B., Zimmermann, M., Marcello, E., Colciaghi,F., Gardoni, F., Intraligi, M., Padovani, A., Buscema, M., 2005. Artificialneural networks allow the use of simultaneous measurements of Alzheimerdisease markers for early detection of the disease. J. Transl. Med. 3, 30.

Di Luca, M., Pastorino, L., Cattabeni, F., Zanardi, R., Scarone, S., Racagni, G.,Smeraldi, E., Perez, J., 1996. Abnormal pattern of platelet APP isoforms inAlzheimer disease and Down syndrome. Arch. Neurol. 53, 1162–1166.

Ferrarese, C., Begni, B., Canevari, C., Zoia, C., Piolti, R., Frigo,M., Appollonio, I.,Frattola, L., 2000. Glutamate uptake is decreased in platelets fromAlzheimer'sdisease patients. Ann. Neurol. 47, 641–643.

Fisk, J.D., Merry, H.R., Rockwood, K., 2003. Variations in case definition affectprevalence but not outcomes of mild cognitive impairment. Neurology 61,1179–1184.

Frisoni, G.B., Padovani, A., Wahlund, L.O., 2003. The diagnosis of Alzheimerdisease before it is Alzheimer dementia. Arch. Neurol. 60, 1023.

Frisoni, G.B., Padovani, A., Wahlund, L.O., 2004. The predementia diagnosis ofAlzheimer disease. Alzheimer Dis. Assoc. Disord. 18, 51–53.

Fukumoto, H., Tennis, M., Locascio, J.J., Hyman, B.T., Growdon, J.H., Irizarry,M.C., 2003. Age but not diagnosis is the main predictor of plasma amyloidbeta-protein levels. Arch. Neurol. 60, 958–964.

Galasko, D., Clark, C., Chang, L., Miller, B., Green, R.C., Motter, R., Seubert,P., 1997. Assessment of CSF levels of tau protein in mildly dementedpatients with Alzheimer's disease. Neurology 48, 632–635.

Ghanbari, K., Ghanbari, H.A., 1998. A sandwich enzyme immunoassay formeasuring AD7C-NTP as an Alzheimer's disease marker: AD7C test. J. Clin.Lab. Anal. 12, 223–226.

Ghiso, J., Frangione, B., 2002. Amyloidosis and Alzheimer's disease. Adv.Drug. Deliv. Rev. 54, 1539–1551.

Growdon, J.H., Selkoe, D.J., Roses, A., 1998. Consensus report of the WorkingGroup on Biological Markers of Alzheimer's disease. Neurobiol. Aging 19,109–116.

Hampel, H., Buerger, K., Zinkowski, R., Teipel, S.J, Goernitz, A., Andreasen,N., Sjoegren, M., DeBernardis, J., Kerkman, D., Ishiguro, K., Ohno, H.,Vanmechelen, E., Vanderstichele, H., McCulloch, C., Moller, H.J., Davies,P., Blennow, K., 2004a. Measurement of phosphorylated tau epitopes in thedifferential diagnosis of Alzheimer disease: a comparative cerebrospinalfluid study. Arch. Gen. Psychiatry 61, 95–102.

Hampel, H., Teipel, S.J., Fuchsberger, T., Andreasen, N., Wiltfang, J., Otto, M.,Shen, Y., Dodel, R., Du, Y., Farlow, M., Moller, H.J., Blennow, K., Buerger,K., 2004b. Value of CSF beta-amyloid1-42 and tau as predictors ofAlzheimer's disease in patients with mild cognitive impairment. Mol.Psychiatry 9, 705–710.

Heinonen, O., Soininen, H., Syrjanen, S., Neittaanmaki, H., Paljarvi, L.,Kosunen, O., Syrjanen, K., Riekkinen Sr., P., 1994. Beta-amyloid proteinimmunoreactivity in skin is not a reliable marker of Alzheimer's disease. Anautopsy-controlled study. Arch. Neurol. 51, 799–804.

Hulstaert, F., Blennow, K., Ivanoiu, A., Schoonderwaldt, H.C., Riemenschnei-der, M., De Deyn, P.P., Bancher, C., Cras, P., Wiltfang, J., Mehta, P.D., Iqbal,K., Pottel, H., Vanmechelen, E., Vanderstichele, H., 1999. Improveddiscrimination of AD patients using beta-amyloid(1-42) and tau levels inCSF. Neurology 52, 1555–1562.

Itoh, N., Arai, H., Urakami, K., Ishiguro, K., Ohno, H., Hampel, H., Buerger, K.,Wiltfang, J., Otto, M., Kretzschmar, H., Moeller, H.J., Imagawa, M., Kohno,H., Nakashima, K., Kuzuhara, S., Sasaki, H., Imahori, K., 2001. Large-scale,multicenter study of cerebrospinal fluid tau protein phosphorylated at serine199 for the antemortem diagnosis of Alzheimer's disease. Ann. Neurol. 50,150–156.

Iwatsubo, T., Hasegawa, M., Ihara, Y., 1994. Neuronal and glial tau-positiveinclusions in diverse neurologic diseases share common phosphorylationcharacteristics. Acta. Neuropathol. (Berl) 88, 129–136.

Larrieu, S., Letenneur, L., Orgogozo, J.M., Fabrigoule, C., Amieva, H., LeCarret, N., Barberger-Gateau, P., Dartigues, J.F., 2002. Incidence andoutcome of mild cognitive impairment in a population-based prospectivecohort. Neurology 59, 1594–1599.

Lewczuk, P., Esselmann, H., Groemer, T.W., Bibl, M., Maler, J.M., Steinacker,P., Otto, M., Kornhuber, J., Wiltfang, J., 2004. Amyloid beta peptides incerebrospinal fluid as profiled with surface enhanced laser desorption/ionization time-of-flight mass spectrometry: evidence of novel biomarkersin Alzheimer's disease. Biol. Psychiatry 55, 524–530.

Linder, J., Nolgard, P., Nasman, B., Back, O., Uddhammar, A., Olsson, T., 1993.Decreased peripheral glucocorticoid sensitivity in Alzheimer's disease.Gerontology 39, 200–206.

Luis, C.A., Loewenstein, D.A., Acevedo, A., Barker, W.W., 2003. Mild cognitiveimpairment: directions for future research. Neurology 61, 438–444.

Maddalena, A., Papassotiropoulos, A., Muller-Tillmanns, B., Jung, H.H., Hegi,T., Nitsch, R.M., Hock, C., 2003. Biochemical diagnosis of Alzheimerdisease by measuring the cerebrospinal fluid ratio of phosphorylated tauprotein to beta-amyloid peptide42. Arch. Neurol. 60, 1202–1206.

Maruyama, M., Arai, H., Sugita, M., Tanji, H., Higuchi, M., Okamura, N.,Matsui, T., Higuchi, S., Matsushita, S., Yoshida, H., Sasaki, H., 2001.Cerebrospinal fluid amyloid beta(1-42) levels in the mild cognitiveimpairment stage of Alzheimer's disease. Exp. Neurol. 172, 433–436.

Masters, C.L., Simms, G., Weinman, N.A., Multhaup, G., McDonald, B.L.,Beyreuther, K., 1985. Amyloid plaque core protein in Alzheimer disease andDown syndrome. Proc. Natl. Acad. Sci. U. S. A. 82, 4245–4249.

Matsushima, H., Shimohama, S., Fujimoto, S., Takenawa, T., Kimura, J., 1995.Changes in platelet phospholipase C protein level and activity inAlzheimer's disease. Neurobiol. Aging. 16, 895–900.

Mayeux, R., Tang, M.X., Jacobs, D.M., Manly, J., Bell, K., Merchant, C., Small,S.A., Stern, Y., Wisniewski, H.M., Mehta, P.D., 1999. Plasma amyloid beta-peptide 1-42 and incipient Alzheimer's disease. Ann. Neurol. 46, 412–416.

Mayeux, R., Honig, L.S., Tang,M.X.,Manly, J., Stern, Y., Schupf, N.,Mehta, P.D.,2003. Plasma A[beta]40 and A[beta]42 and Alzheimer's disease: relation toage, mortality, and risk. Neurology 61, 1185–1190.

Mehta, P.D., Pirttila, T., Mehta, S.P., Sersen, E.A., Aisen, P.S., Wisniewski, H.M.,2000. Plasma and cerebrospinal fluid levels of amyloid beta proteins 1-40 and1-42 in Alzheimer disease. Arch. Neurol. 57, 100–105.

Montine, T.J., Sidell, K.R., Crews, B.C., Markesbery, W.R., Marnett, L.J.,Roberts 2nd, L.J., Morrow, J.D., 1999. Elevated CSF prostaglandin E2levels in patients with probable AD. Neurology 53, 1495–1498.

Morris, J.C., Storandt, M., Miller, J.P., McKeel, D.W., Price, J.L., Rubin, E.H.,Berg, L., 2001. Mild cognitive impairment represents early-stage Alzheimerdisease. Arch. Neurol. 58, 397–405.

Motter, R., Vigo-Pelfrey, C., Kholodenko, D., Barbour, R., Johnson-Wood, K.,Galasko, D., Chang, L., Miller, B., Clark, C., Green, R., 1995. Reduction ofbeta-amyloid peptide42 in the cerebrospinal fluid of patients with Alzheimer'sdisease. Ann. Neurol. 38, 548–643.

Mufson, E.J., Chen, E.Y., Cochran, E.J., Beckett, L.A., Bennett, D.A.,Kordower, J.H., 1999. Entorhinal cortex beta-amyloid load in individualswith mild cognitive impairment. Exp. Neurol. 158, 469–490.

Nestor, P.J., Scheltens, P., Hodges, J.R., 2004. Advances in the early detection ofAlzheimer's disease. Nat. Med. S34–S41.

Padovani, A., Borroni, B., Colciaghi, F., Pastorino, L., Archetti, S., Cottini, E.,Caimi, L., Cattabeni, F., Di Luca, M., 2001a. Platelet amyloid precursorprotein forms in AD: a peripheral diagnostic tool and a pharmacologicaltarget. Mech. Ageing Dev. 122, 1997–2004.

Padovani, A., Pastorino, L., Borroni, B., Colciaghi, F., Rozzini, L., Monastero,R., Perez, J., Pettenati, C., Mussi, M., Parrinello, G., Cottini, E., Lenzi, G.L.,Trabucchi, M., Cattabeni, F., Di Luca, M., 2001b. Amyloid precursor proteinin platelets: a peripheral marker for the diagnosis of sporadic AD. Neurology57, 2243–2248.

Padovani, A., Borroni, B., Colciaghi, F., Pettenati, C., Cottini, E., Agosti, C.,Lenzi, G.L., Caltagirone, C., Trabucchi, M., Cattabeni, F., Di Luca, M.,2002. Abnormalities in the pattern of platelet amyloid precursor proteinforms in patients with mild cognitive impairment and Alzheimer disease.Arch. Neurol. 59, 71–75.

Palmert, M.R., Usiak, M., Mayeux, R., Raskind, M., Tourtellotte, W.W., Younkin,S.G., 1990. Soluble derivatives of the beta amyloid protein precursor in

80 B. Borroni et al. / European Journal of Pharmacology 545 (2006) 73–80

cerebrospinal fluid: alterations in normal aging and in Alzheimer's disease.Neurology 40, 1028–1034.

Petersen, R.C., Smith, G.E., Waring, S.C., Ivnik, R.J., Tangalos, E.G., Kokmen,E., 1999. Mild cognitive impairment: clinical characterization and outcome.Arch. Neurol. 56, 303–308.

Petersen, R.C., Doody, R., Kurz, A., Mohs, R.C., Morris, J.C., Rabins, P.V.,Ritchie, K., Rossor, M., Thal, L., Winblad, B., 2001. Current concepts inmild cognitive impairment. Arch. Neurol. 58, 1985–1992.

Pirttila, T.,Mattinen, S., Frey, H., 1992. The decrease ofCD8-positive lymphocytesin Alzheimer's disease. J. Neurol. Sci. 107, 160–165.

Pratico, D., Delanty, N., 2000. Oxidative injury in diseases of the central nervoussystem: focus on Alzheimer's disease. Am. J. Med. 109, 577–585.

Pratico, D., M.Y. Lee, V., Trojanowski, J.Q., Rokach, J., Fitzgerald, G.A., 1998.Increased F2-isoprostanes in Alzheimer's disease: evidence for enhancedlipid peroxidation in vivo. FASEB J. 12, 1777–1783.

Pratico, D., Clark, C.M., Lee, V.M., Trojanowski, J.Q., Rokach, J., FitzGerald, G.A., 2000. Increased 8,12-iso-iPF2alpha-VI in Alzheimer's disease: correlationof a noninvasive index of lipid peroxidation with disease severity. Ann.Neurol.48, 809–812.

Riemenschneider, M., Schmolke, M., Lautenschlager, N., Guder, W.G.,Vanderstichele, H., Vanmechelen, E., Kurz, A., 2000. Cerebrospinal beta-amyloid (1-42) in early Alzheimer's disease: association with apolipoproteinE genotype and cognitive decline. Neurosci. Lett. 284, 85–88.

Rosenberg, R.N., Baskin, F., Fosmire, J.A., Risser, R., Adams, P., Svetlik, D.,Honig, L.S., Cullum, C.M., Weiner, M.F., 1997. Altered amyloid proteinprocessing in platelets of patients with Alzheimer disease. Arch. Neurol. 54,139–144.

Schoonenboom, N.S., Pijnenburg, Y.A., Mulder, C., Rosso, S.M., Van Elk, E.J.,Van Kamp, G.J., Van Swieten, J.C., Scheltens, P., 2004. Amyloid beta(1-42)and phosphorylated tau in CSF as markers for early-onset Alzheimerdisease. Neurology 62, 1580–1584.

Sjogren, M., Davidsson, P., Tullberg, M., Minthon, L., Wallin, A., Wikkelso, C.,Granerus, A.K.,Vanderstichele,H., Vanmechelen, E., Blennow,K., 2001. Bothtotal and phosphorylated tau are increased in Alzheimer's disease. J. Neurol.Neurosurg. Psychiatry 70, 624–630.

Sjogren, M., Andreasen, N., Blennow, K., 2003. Advances in the detection ofAlzheimer's disease—use of cerebrospinal fluid biomarkers. Clin. Chim.Acta 332, 1–10.

Skoog, I., Davidsson, P., Aevarsson, O., Vanderstichele, H., Vanmechelen, E.,Blennow, K., 2003. Cerebrospinal fluid beta-amyloid 42 is reduced beforethe onset of sporadic dementia: a population-based study in 85-year-olds.Dement. Geriatr. Cogn. Disord. 15, 169–176.

Smith, R.P., Higuchi, D.A., Broze Jr., G.J., 1990. Platelet coagulation factorXIa-inhibitor, a form of Alzheimer amyloid precursor protein. Science 248,1126–1128.

Soininen, H., Syrjanen, S., Neittaanmaki, H., Miettinen, R., Paljarvi, L.,Syrjanen, K., Beyreuther, K., Riekkinen, P., 1992. Amyloid beta-proteindeposition in skin of patients with dementia. Lancet 339, 245.

Sunderland, T., Linker, G., Mirza, N., Putnam, K.T., Friedman, D.L., Kimmel, L.H.,Bergeson, J., Manetti, G.J., Zimmermann, M., Tang, B., Bartko, J.J., Cohen,R.M., 2003. Decreased beta-amyloid1-42 and increased tau levels in cere-brospinal fluid of patients with Alzheimer disease. JAMA. 289, 2094–2103.

Tamaoka, A., Odaka, A., Ishibashi, Y., Usami, M., Sahara, N., Suzuki, N., Nukina,N., Mizusawa, H., Shoji, S., Kanazawa, I., 1994. APP717 missense mutationaffects the ratio of amyloid beta protein species (Abeta 1-42/43 and a beta 1-40)in familial Alzheimer's disease brain. J. Biol. Chem. 269, 32721–32724.

Tamaoka, A., Fukushima, T., Sawamura, N., Ishikawa, K., Oguni, E., Komatsuzaki,Y., Shoji, S., 1996. Amyloid beta protein in plasma from patients with sporadicAlzheimer's disease. J. Neurol. Sci. 141, 65–68.

The Ronald and Nancy Reagan Research Institute of the Alzheimer's Associationand theNational Institute onAgingWorkingGroup, 1998. Consensus report ofthe Working Group on: molecular and biochemical markers of Alzheimer'sdisease. Neurobiol. Aging. 109–116.

VanNostrand,W.E.,Wagner, S.L., Haan, J., Bakker, E., Roos, R.A., 1992.Decreasedlevels of soluble amyloid beta protein precursor in cerebrospinal fluid of liveAlzheimer disease patients. Proc. Natl. Acad. Sci. U. S. A. 89, 2551–2555.

Vanderstichele, H., Van Kerschaver, E., Hesse, C., Davidsson, P., Buyse, M.A.,Andreasen, N., Minthon, L., Wallin, A., Blennow, K., Vanmechelen, E., 2000.Standardization of measurement of beta-amyloid(1-42) in cerebrospinal fluidand plasma. Amyloid 7, 245–258.

Vanderstichele, H., De Meyer, G., Andreasen, N., Kostanjevecki, V., Wallin, A.,Olsson, A., Blennow, K., Vanmechelen, E., 2005. Amino-truncated beta-amyloid42 peptides in cerebrospinal fluid and prediction of progression ofmild cognitive impairment. Clin. Chem. 51, 1650–1660.

Von Strauss, E., Viitanen, M., De Ronchi, D., Winblad, B., Fratiglioni, L., 1999.Aging and the occurrence of dementia: findings from a population-basedcohort with a large sample of nonagenarians. Arch. Neurol. 56, 587–592.

Wiltfang, J., Esselmann, H., Bibl, M., Smirnov, A., Otto, M., Paul, S., Schmidt, B.,Klafki, H.W., Maler, M., Dyrks, T., Bienert, M., Beyermann, M., Ruther, E.,Kornhuber, J., 2002. Highly conserved and disease-specific patterns ofcarboxyterminally truncated Abeta peptides 1-37/38/39 in addition to 1-40/42in Alzheimer's disease and in patients with chronic neuroinflammation.J. Neurochem. 81, 481–496.

Zoia, C., Cogliati, T., Tagliabue, E., Cavaletti, G., Sala, G., Galimberti, G., Rivolta,I., Rossi, V., Frattola, L., Ferrarese, C., 2004. Glutamate transporters inplatelets: EAAT1 decrease in aging and in Alzheimer's disease. Neurobiol.Aging. 25, 149–157.

Zubenko, G.S., Wusylko, M., Cohen, B.M., Boller, F., Teply, I., 1987. Familystudy of platelet membrane fluidity in Alzheimer's disease. Science 238,539–542.