Advances in the detection of Alzheimer's disease—use of cerebrospinal fluid biomarkers

Clinica Chimica Acta 332 (2003) 1 – 10 www.elsevier.com/locate/clinchim

Review

Advances in the detection of Alzheimer’s disease—use of cerebrospinal fluid biomarkers Magnus Sjo¨gren a,*, Niels Andreasen b, Kaj Blennow a a

Institute of Clinical Neuroscience, Go¨teborg University, SE 431 80 Go¨teborg, Sweden b Karolinska Institute, Neurotec, Huddinge University Hospital, Huddinge, Sweden

Received 28 October 2002; received in revised form 10 February 2003; accepted 10 February 2003

Abstract The diagnosis of Alzheimer’s disease (AD) is still made by excluding other disorders with a similar clinical picture. In addition, an analysis of symptoms and signs, blood analyses and brain imaging are the major ingredients of the clinical diagnostic work-up. However, the sensitivity of a clinical diagnosis using these instruments is unsatisfactory and disease markers with high sensitivity and specificity for AD would be a welcome supplement. Ideally, such markers should reflect the pathophysiological mechanisms of AD, that is, according to the currently predominant hypothesis mismetabolism of h-amyloid and neurofibrillary degeneration. Among several, we have focused on three candidates that have been suggested to fulfil the requirements for biomarkers of AD: h-amyloid42 (Ah42), total tau (T-tau) and tau phosphorylated at various epitopes (P-tau). The cerebrospinal fluid (CSF) levels of these proteins reflect the metabolism of these proteins in the central nervous system. Only published articles using established ELISA methods for the quantification of these markers in CSF and preferably also presenting sensitivity and specificity figures have been included in this review. The number of patients included in the different studies varies; some having included only a few patients. Furthermore, diagnostic criteria vary and clinicopathological studies are scarce. However, there are some large studies, including even minor studies, and most have found reduced CSF levels of Ah42 and increased CSF levels of T-tau in AD. The sensitivity and specificity of these measures are high for separation of AD patients from controls, but their specificity against other dementias is moderate. It increases if P-tau is added. An increasing number of studies suggest that supplementary use of these CSF markers, preferably in combination, adds to the accuracy of an AD diagnosis. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Alzheimer’s disease; h-Amyloid; Biochemical markers; Cerebrospinal fluid; Diagnosis; Mild cognitive impairment; Tau

1. Introduction Alzheimer’s disease (AD) is a major health threat and one of the most costly diseases in modern society. * Corresponding author. Tel.: +46-31-343-2382; fax: +46-31776-9055. E-mail address: [email protected] (M. Sjo¨gren).

Estimates indicate that approximately 4 million people in the United States suffer from AD. The disorder, which affects men and women equally, is characterized by progressive deterioration of cognitive functions, such as memory, language and visuospatial orientation. Associated symptoms are mood and behavioural changes. The prognosis is poor with no cure available. AD imposes a heavy burden on the caregivers in the

0009-8981/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0009-8981(03)00121-9

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families and in the health care system. Society’s total cost for care of patients with dementia is substantial. Estimates have suggested that the cost for a country such as Sweden is about 0.4 billion Euro per million inhabitants [1]. A rapid increase in the elderly population is expected, especially in the developing countries, and this will lead to a further increase in the cost, if no cure is found. The cause of AD is unknown in most cases, but mutations in a few underlying genes have been identified in familial AD [2]. Underlying neuropathological changes in AD are the accumulation of senile plaques (SPs) and neurofibrillary tangles (NFTs). SPs are made up mainly of h-amyloid, especially the 42-amino-acid isoform, h-amyloid 42 (Ah42) [3]. The major constituent of NFTs is a cytoskeleton-associated protein called tau, which is hyperphosphorylated in NFTs [4]. The golden standard of diagnosis is the identification of typical neuropathological changes in the brain of a patient who has suffered from clinical AD. In clinical routine, the diagnosis of AD, as outlined in the NINDSADRDA criteria [5], is based on clinical and neuropsychological examinations, identification of typical symptoms of AD and exclusion of other known causes of dementia. Studies have shown that the accuracy of the clinical diagnosis is between 65% and 90%. Higher accuracy is achieved at academic centres with special interest in AD. This is partly due to the fact that these centres include patients in the later stages of the disease who have been followed for several years before the confirming autopsy [6– 8]. The accuracy of the clinical diagnosis at the primary care level and in general hospitals is probably even lower, especially in the early stages of the disease when the symptoms are indistinct. In view of this, the need for specific AD markers is great. According to a proposal of a consensus group on molecular and biochemical markers of AD [9], an ideal marker of AD should be able to detect a fundamental feature of neuropathology and should be validated against neuropathologically confirmed cases. Furthermore, its sensitivity for detection of AD as well as its specificity for discrimination of AD from other dementia disorders should exceed 80%. A marker for AD should also be reliable, reproducible, noninvasive, simple to perform in clinical routine and inexpensive. As the cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the brain [10], biochemical changes in the brain, for instance,

those caused by accumulation of SPs and NFTs, will lead to a change in the biochemistry of the CSF [11]. CSF is therefore an appropriate source of biochemical markers for AD. There are three candidates that have been suggested to fulfil the requirements stated by the consensus group [9]: total tau protein (T-tau), Ah42 and tau phosphorylated at AD-specific epitopes (P-tau). 1.1. T-tau Tau is a microtubule-associated protein which is located mainly in neuronal axons. By binding to microtubules, it promotes the stability and function of these. In the normal human brain, six different isoforms of tau are found, all of which have numerous phosphorylation sites [12]. As tau is a major constituent of NFTs, CSF T-tau has been suggested as a marker for AD. Using monoclonal antibodies that detect all isoforms of tau independent of degree of phosphorylation, enzyme-linked immunosorbent assays (ELISAs) have been developed that measure the T-tau levels in CSF [13 – 15] (Fig. 1). Using these ELISAs, more than 50 studies have consistently demonstrated a moderate to marked increase in CSF T-tau as well as high sensitivity and specificity of CSF-tau in AD patients when compared with controls (Table 1). So far, CSF from about 2400 AD patients and 1250 controls has been investigated in this way (Table 1). The mean degree of increase is about 300% in AD compared with controls. The high sensitivity and specificity make CSF T-tau a good candidate for the designation biochemical marker for AD, or AD biomarker. However, high levels of T-tau in the CSF have also been found in a proportion of cases with other dementia disorders, such as frontotemporal dementia [16,17] and Lewy body dementia [18], but in several other disorders, for example, alcohol dementia, Parkinson’s disease and depression, the CSF levels of T-tau seem to be normal and only occasionally increased [14,17,19,20]. What does an increase in CSF T-tau reflect? Few studies have directly investigated this, but it has been suggested that the CSF T-tau levels reflect the degree of neuronal (especially axonal) degeneration and damage [14]. Some evidence for this has been found, for instance, a transient increase in CSF T-tau after acute stroke, with a positive correlation between CSF T-tau

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Fig. 1. (A) Principles for an ELISA specific to total tau (T-tau) in which three monoclonal antibodies (AT120, HT7 and BT2) are used. All these antibodies recognize tau irrespective of its phosphorylation state and are specific to epitopes outside the alternatively spliced exons. (B) A schematic picture of human tau protein with the six isoforms. Alternatively spliced exons (exons 2, 3 and 10) are shown. At top, the smallest tau isoform (352 amino acids) containing three repeat domains and, at bottom, the largest tau isoform (441 amino acids) containing four repeat domains (including exon 10) and two extra domains from exons 2 and 3.

and infarct size as measured by computerized tomography [21], a very marked increase in CSF T-tau in Creutzfeldt – Jakob’s disease [22], and a correlation between premortem CSF T-tau levels and the postmortem density of neurofibrillary tangles in the brain [23]. Indirect evidence is that, in AD and controls, there is a positive correlation between the CSF levels of Ttau, GAP-43 and amyloid precursor protein (APP), all proteins located in the axon of neurons [24]. 1.2. Ab42 The central protein in SPs is Ah42. It is produced and secreted from human cells as a result of normal cellular processing of the larger transmembrane protein APP [25] (Fig. 2). In this processing, APP is cleaved in several steps and Ah is produced. In, AD, APP is first cleaved by an enzyme called h-secretase, which results in the release of a large N-terminal fragment called h-secretase-cleaved soluble APP. In

a second step, APP is cleaved by the g-secretase complex, which results in the release of free Ah (Fig. 2). In this processing, various isoforms of Ah, for example, Ah42, are produced; all of which are secreted into the CSF. Using four different ELISA methods that are specific to Ah42 [26 – 29], more than 30 studies have consistently demonstrated a moderate to marked decrease in CSF Ah42 in AD. The principle for the ELISA that is most commonly used to measure Ah42 in CSF, INNOTESTk h-AMYLOID(1 – 42) [29], is shown in Fig. 2. There are 13 studies, including a total of about 600 AD cases and 450 controls, in which sensitivity and specificity figures have been given or can be calculated from graphs (Table 2). These studies show that, for CSF Ah42, the mean sensitivity for discrimination between AD and normal aging is approximately 86%, while the specificity is approximately 91% and the mean level of decrease in AD patients compared with controls is about 50% (Table 2).

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Table 1 Performance of CSF total tau Country

Study

EU Japan EU

Blennow, 1995 Arai, 1995 Jensen, 1995

EU EU EU

Riemenschneider, 1996 Ro¨sler, 1996 Andreasen, 1998 (JNNP)

Japan Japan EU EU Japan Japan EU EU Japan EU

Arai, 1998 Kanai, 1998 (Ann. Neurol.) Kurz, 1998 (Alz. Dis.) Mecocci, 1998 (Alz. Dis.) Nishimura, 1998 (Meth. F. Exp. Cl. Ph.) Shoiji, 1998 (J. Neurol. Sci.) Tapiola, 1998 (Neurology) Vanderstichele, 1998 (Progr. AD/PD) Morihara, 1998 (Psy. Clin. Neurosci.) Andreasen, 1999 (Neurology)

EU

Burger, 1999 (Neurosci. Lett.)

EU EU EU + USA Japan EU Japan USA

Green, 1999 (Neurosci. Lett.) Hampel, 1999 (Brain Res.) Hulstaert, 1999 (Neurology) Ishiguro, 1999 (Neurosci. Lett.) Martinez, 1999 (Brain Res.) Morikawa, 1999 (Alc. Clin. Exp. Res.) Kahle, 2000 (Neurology)

Japan EU EU

Kanemaru, 2000 (Neurology) Sjo¨gren, 2000 (J. Neural Transm.) Sjo¨gren, 2000 (Neurology)

EU

Gottfries, 2001 (J. Ger. Psy. Neurol.)

Japan EU Japan EU EU EU Japan EU EU EU EU Sum/mean

Itoh, 2001 (Ann. Neurol.) Kapaki, 2001 (JNNP) Maruyama, 2001 (Exp. Neurol.) Ro¨sler, 2001 (J. Neural Transm.) Sjo¨gren, 2001 (Dementia) Buerger, 2002 (Arch. Neurol.) Hu, 2002 (Am. J. Pathol.) Briani, 2002 (J. Neural Transm.) Mulder, 2002 (J. Neural Transm.) Sjo¨gren, 2002 (Dementia) Riemenschneider, 2002 (Neurology)

AD number

AD sensitivity

Percentage increase

Control number

Control specificity

44 70 15

84 100 95

283 858 661

31 19 22

97 100 100

11 16 43

95 88 95

446 n.g. 419

19 10 18

95 100 94

11 69 93 40 29 163

100 89 40 89 52 66

398 443 226 442 205 227

17 41 36 15 65

100 98 97 73 83

55 81 81 11 407

31 58 90 91 93

214 179 178 312 304

34 33 15 14 93

97 88 67 100 86

15 23 17 25 150 36 10 36 30 5 24 60 21 21 43

87 83 76 80 79 97 100 92 63 80 83 79 76 57 95

212 n.a. 405 231 218 n.g. 544 505 247 n.g. 400 242 200 186 204

25 22 9 19 100 20 10 23 16

76 46 100 85 70 50 90 95 75

19 32 18

95 82 85

n.a.

n.a.

303 358 351 320 237 n.g. 226 293 223 269 355 324.3

95 47 15 49 12 21 56 17 20 17 40 1184

236 38 54 27 47 80 52 9 20 19 74 2411

85 90 87 89 77 81.3 79 67 90 84 95 81.6

Comment

Values as tau/total protein; 8 FAD, 7 sporadic AD

Community-based patient sample Mixed AD/VAD

Neurological controls

Controls including depression < 70 years >70 years

‘‘Non-AD’’ controls

Neuropathological AD

Early onset AD Late onset AD Compared with reference values Approx. sens/spec

92 93 100 91 100 88 90 94 98 88.4

Controls with lumbago

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Fig. 2. (A) A schematic picture of amyloid precursor protein (APP) and the generation of free h-amyloid (Ah). APP is cleaved by two proteases, first h-secretase then g-secretase. Ah is thereby released and secreted into the CSF. (B) Principles for an ELISA specific to Ah42 in which the capture antibody (21F11) is specific to the C terminus of Ah (Ah42) and the detection antibody (3D6) specifically recognizes the N terminus of Ah (Ah1).

On the other hand, the specificity for discrimination of AD from other disorders is moderate. Low levels of Ah42 in CSF have, for example, been found in Lewy body dementia [18,30], a disorder also characterized by the presence of SPs. Low levels have also been found in a small percentage of patients with frontotemporal dementia and vascular dementia [31,32] and also in

Creutzfeldt – Jakob’s disease [33,34] and amyotrophic lateral sclerosis [35]. These studies question the putative relation between low CSFAh42 levels and the accumulation of SPs. There are several possible causes of low CSF-Ah42 levels, for example, axonal degeneration [35,36] and entrapment in narrow interstitial and subarachnoid drainage pathways [37].

Table 2 Performance of CSF Ah42 Country

Study

EU EU

Vanderst, 1998 (Progr. AD/PD) Andreasen, 1999 (Arch. Neurol.)

EU EU + USA Japan EU EU EU EU EU EU EU EU Sum/mean

Andreasen, 1999 (Neurosci. Lett.) Hulstaert, 1999 (Neurology) Kanemaru, 2000 (Neurology) Otto, 2000 (Neurology) Sjo¨gren, 2000 (J. N. Transm.) Kapaki, 2001 (JNNP) Ro¨sler, 2001 (J. Neural Transm.) Briani, 2002 (J. Neural Transm.) Mulder, 2002 (J. Neural Transm.) Sjo¨gren, 2002 (Dementia) Riemenschneider, 2002 (Neurology)

AD number

AD sensitivity

81 53

81 92

16 150 24 14 60 38 27 9 20 19 74 585

88 78 96 93 93 76 78 55 100 100 89 86.1

Percentage decrease 75 42 72 57 40 40 49 51 48 73 46 42 37 51.7

Control number 51 21 15 100 19 20 32 47 49 17 20 17 40 448

Control specificity 80 95 80 81 95 95 n.g. 85 100 94 95 94 95 90.8

Comment

Community-based sample

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1.3. P-tau Tau is normally in a phosphorylated state. Over 70 phosphorylation sites are found on the human tau molecule (Fig. 3) and, in AD, tau is usually in a hyperphosphorylated state. In AD, this hyperphosphorylation involving certain epitopes on the tau molecule has the consequence that tau loses its ability to promote microtubule assembly and stability, which in turn leads to cytoskeleton instability and diminished transport ability [38,39]. A consequence of this is aggregation of tau with subsequent formation of NFTs [12]. Several ELISAs have been developed that use monoclonal antibodies directed toward sites that are phosphorylated in AD. The principle for one of these ELISAs, INNOTESTk PHOSPHO-TAU(181P), which measures tau phosphorylated at threonine 181 (P-Tau181), is given in Fig. 3 [40]. Other ELISAs identify tau phosphorylated at the epitopes threonine 181 and 231 (P-tau181 + 231) [14], threonine 231 and serine 235 (P-tau231 + 235) [41], serine 199 (P-tau199) [41], threonine 231 (P-tau231) [42] and serine 396 and 404 (P-tau396 + 404) [43]. All these assays have shown increased CSF levels of P-tau in AD patients compared with controls (Table 3). The sensitivity of CSF P-tau for discrimination

between AD and normal aging is about the same or slightly lower as that of CSF T-tau, that is, about 75%. Interestingly, the specificity of CSF P-tau for discrimination of AD from other dementias seems to be higher than those of CSF T-tau and CSF Ah42. Normal CSF levels of P-tau have been found in vascular dementia, frontotemporal dementia [44] and Lewy body dementia [45], which suggests that the above ELISAs may help to discriminate between AD and these dementias. In addition, while there is a marked increase in CSF T-tau after acute stroke, the CSF P-tau does not change [46]. This suggests that the origin of increased CSF P-tau levels is more closely related to AD pathology, for instance, the formation of NFTs.

2. Combination of CSF markers for AD The rationale for using the CSF levels of T-tau, Ah42 and P-tau in combination to detect AD is very clear. Because the concentrations of any one of these substances is believed to reflect central pathogenetic processes in the disorder, that is, according to the leading hypothesis on the development of AD, the amyloid cascade hypothesis, the combination might

Fig. 3. (A) A schematic picture of the largest tau isoform (441 amino acids), with phosphorylation sites, either threonine or serine. (B) Principles for an ELISA specific to phospho-tau (P-Thr181) in which the capture antibody (HT7) recognizes all forms of tau and the detection antibody (AT180) specifically recognizes tau phosphorylated at threonine 181.

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Table 3 Performance of CSF phospho-tau Country

Study

P-tau epitope

EU

Blennow, 1995 (Mol. Chem. Neurop.) Vanmechelen, 2000 (Neurosci. Lett.) Parnetti, 2001 (Neurol. Sci.) Sjo¨gren, 2001 (JNNP) Sjo¨gren, 2002 (Dementia) Ishiguro, 1999 (Neurosci. Lett.) Ishiguro, 1999 (Neurosci. Lett.) Itoh, 2001 (Ann. Neurol.) Kohnken, 2000 (Neurosci. Lett.) Buerger, 2002 (Arch. Neurol.) Hu, 2002 (Am. J. Pathol.)

P-Thr181 + P-Thr231

40

88

348

31

97

P-Thr181

41

44

148

17

94

P-Thr181

80

84

n.g.

40

88

P-Thr181 P-Thr181

60 19

37 58

145 164

17 17

94 94

P-Thr231 + P-Ser235

36

53

n.g.

20

100

P-Ser199

36

94

n.g.

20

80

P-Ser199

236

85

317

95

84

P-Thr231

27

85

n.g.

31

97

P-Thr231

82

100

n.g.

21

91

P-Ser396 + 404

52

83

346

56

98

73.7

244.7

EU EU + USA EU EU Japan Japan Japan USA EU USA + China Sum/mean

AD number

709

result in increased sensitivity and specificity. In fact, some large studies have shown that both sensitivity and specificity increase when, for instance, CSF T-tau and CSF Ah42 are used in combination instead of being used alone [31,47 – 49]. Moreover, in a community-based setting, the sensitivity for AD was more than 90%, when combinations of the above CSF markers for AD were used in routine clinical chemistry analyses. The sensitivity and specificity figures were based on the values for all consecutive patients admitted for investigation of cognitive disturbances during 1 year [49].

AD sensitivity

Percentage increase

Control number

365

Control specificity

Comment

‘‘Non-AD’’ controls ‘‘Non-AD’’ controls

92.4

low CSF levels of Ah42 in patients with mild cognitive impairment (MCI) who later developed AD [50,54,55]. Increased CSF levels of T-tau were also found to discriminate, with high sensitivity and specificity, MCI patients whose disturbances later progressed to AD from the others [54]. Other studies have also found increased CSF levels of P-tau in a high proportion of MCI cases [54,55]. These findings suggest that all three CSF markers may be of use in the clinical identification of AD in the very early phases of the disease and thus facilitate early intervention.

4. CSF markers for AD in clinical practice 3. CSF markers in mild cognitive impairment and in early AD High CSF levels of T-tau and low CSF levels of Ah42 in the early stages of AD have been found in several studies [31,47,50 – 53]. For more severely demented AD cases, the sensitivity figures are 80– 90%, suggesting that the two CSF markers are workable in the early stages of the disease process. Several studies have also found high CSF levels of T-tau and

Attention has been focused on finding one single marker for AD. This seems possible only if the marker is related to a pathogenetic step that is unique to AD. However, neuronal and synaptic degeneration is not only found in AD but in most chronic degenerative disorders of the brain. Similarly, deposition of Ah is not specific to AD, but also found in normal aging, dementia pugilistica, Lewy body dementia and after acute brain trauma, while formation of PHF into tangles

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may occur in normal aging, dementia pugilistica, myotonic dystrophy and hereditary frontotemporal dementia [56 – 58]. This reduces the likelihood of finding one single biochemical marker for AD. Instead, the combination of several CSF biochemical markers (e.g. CSF T-tau, CSF Ah42 and CSF P-tau) could be used in conjunction with other diagnostic methods to enable high accuracy of the clinical diagnosis. Today, the CSF markers T-tau, P-tau and Ah42, when used as adjuncts to the clinical diagnosis, have the potential to help to differentiate AD from some problematic differential diagnoses, especially normal aging, depressive pseudo-dementia, Parkinson’s disease, progressive supranuclear palsy and alcoholic dementia.

Acknowledgements This study was supported by grants from Alzheimerfonden; Bohuslandstingets FoU fond; Fredrik och Ingrid Thurings Stiftelse; Martina och Wilhelm Lundgrens Stiftelse; Stiftelsen fo¨r Gamla Tja¨narinnor; Stiftelsen Handlanden Hjalmar Svenssons Forskningsfond; and the Swedish Medical Research Council (Project nos. 11560 and 12103).

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