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Neurochemical Research, Vol. 30, No. 12, December 2005 (Ó 2005), pp. 1501–1510 DOI: 10.1007/s11064-005-8827-7

Problems Associated with Biological Markers of Alzheimer’s Disease* H. J. Frey,1,4 K. M. Mattila,1,3 M. A. Korolainen,2 and T. Pirttila¨2 (Accepted August 9, 2005)

The etiopathogenesis of Alzheimer’s disease (AD) is still unclear, although clinical diagnostic criteria exist and the neuropathology of AD has been studied in great detail during the last 20 years. The present study addresses certain problems in the search for biological markers for the diagnosis, as well as in the follow-up of the course of AD and its differential diagnosis and reports some of our own observations in comparison with other studies. These include protein, genetic and neuroimaging markers. The definitions of biological markers and search strategies are also discussed. KEY WORDS: Alzheimer’s disease; biological markers; cerebrospinal fluid; proteomics; proteins; immunology; genetics; imaging; blood.

primary cause(s) and pathogenesis of the condition are still largely unknown. The pathological hallmarks of AD are: (1) SPs, (2) NFTs, and (3) congophilic angiopathy, i.e., depositions of b-amyloid (Ab) within vessel walls. These protein aggregates are also observable in the brain during normal aging, but are more abundant in AD. The core of SP is deposited Ab protein, which is a 39–43 amino acid peptide with a molecular mass of 4 kDa (2–4). It is proteolytically derived from a parent protein, the amyloid precursor protein (APP) (4). Other protein components of SPs include serum amyloid P (5), a1-antichymotrypsin (6), sulphated glycosaminoglycans (7), a1-antitrypsin (8), apolipoprotein E (9), and the neurotrophic factor midkine (10). The principal feature of NFTs is the intraneuronal accumulation of insoluble paired helical filaments (PHFs). As nerve cells die, PHFs become extracellular (ghost tangles). The abnormally fosforylated microtubulule-associated tau is the main constituent of PHF (11). The development of neurofibrillary pathology follows a characteristic distribution pattern that was first described by Braak and Braak (12).

INTRODUCTION Molecular Pathology of Alzheimer’s disease The initial report by Alzheimer in 1907 (1) on a middle-aged woman afflicted with a dementing brain disease reported the presence of senile plaques (SPs) and aberrant fibrillary structures referred to as neurofibrillary tangles (NFTs) in the brain tissue. The entity has subsequently become known as Alzheimer’s disease (AD) and is today the most frequent form of dementia, including sporadic and familial disease as well as early and late onset types. Except for the rare inherited early onset disease forms, the * Special issue dedicated to Dr. Simo S. Oja. 1 Brain Research Center, Medical School, University of Tampere, Tampere, Finland. 2 Department of Neurology, University of Kuopio and Kuopio University Hospital, Kuopio, Finland. 3 Centre for Laboratory Medicine, Tampere University Hospital, Tampere, Finland. 4 Address reprint requests to: Prof. Harry Frey, Brain Research Center, Medical School, Building B, University of Tampere, FIN-33014 Tampere, Finland. Tel: +358-3-3551-6735; Fax: +358-3-3551-6170; E-mail: harry.frey@uta.fi

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Frey, Mattila, Korolainen, and Pirttila¨

Pathological Diagnosis of AD Two histopathologic guidelines for the pathological diagnosis of AD are widely used. The Consortium to Establish a Registry for Alzheimer’s Disease (CERAD) (13) emphasizes the presence of senile plaques and The National Institute on Aging and Reagan Institute Working Group (14) also places emphasis on NFT in medial temporal lobe structures and the neocortex. Although SPs and neurofibrillary pathology are considered the core pathology in AD, many recent studies have indicated that the pathogenesis of AD is a complex and heterogeneous process. Gliosis, chronic inflammatory reactions, excitotoxic damage and oxidative stress all appear to contribute to progressive cell death in the AD brain (15). Many studies have shown that the use of clinical (16) criteria predict the presence of AD pathology with high accuracy. However, the sensitivity remains low (31–52%). The latter may be due to the frequent existence of AD-type pathology in cognitively healthy subjects and other dementias (17,18). A recent population-based study reported that 33% of non-demented subjects had densities of neocortical neuritic plaques indicating probable or definite AD and 34% had some degree of neurofibrillary pathology (19). AD pathology is also common in other dementias such as Lewy body dementia and vascular dementia (20,21). Definition of a Biological Disease Marker Biological markers can be defined as parameters, ascertained by measure and quantity, which serve as indicators for health and disease. They primarily identify the existence or the absence of a given disease or confirm the risk of developing the disease. The biological markers can be classified, e.g., according to their relation to the illness: they can be primary (specific) or secondary to the disease, or they can

simply be epiphenomenal in nature. Sensitivity, specificity and ease of use are the most important factors that ultimately define the usefulness of a marker for diagnostic purposes. A variety of tools, including biochemical, molecular genetic and neuroimaging studies, have been used in the search for putative markers for AD. Two basic approaches have been employed. Hypothesis-driven approaches have focused on the characteristic pathological features of AD whereas discovery-type approaches have used modern technologies such as different proteomic methodologies. One major problem in the development of disease markers for AD is that clinically examined control populations often include subjects with AD-type pathology, given the frequent brain pathology in asymptomatic individuals as shown in neuropathological studies. Longitudinal, neuropathologically examined control series are largely nonexistent. The other problem is the heterogeneity of associated brain changes in AD patients. It is possible that no single marker exists that could be used for the early diagnosis, progression and measurement of treatment effects. More likely there are markers that reflect the presence of the disease and others that reflect the progression of the disease pathology.

PROTEIN MARKERS ASSOCIATED WITH AD A wide variety of different proteins such as inflammatory markers, markers of oxidative stress, apolipoproteins, and markers of neuronal degeneration in blood and cerebrospinal fluid (CSF) have been examined in studying AD (Table I). However, most of these studies have yielded negative results. Because the composition of CSF is likely to closely reflect that of the brain intercellular spaces, the search for a biological marker of AD has mainly focused on measurements of CSF constituents.

Table I. Examples of Possible Biological Markers for AD 1 2 3 4 5 6 7 8 9

Neurofibrillary pathology; Tau-protein, phosphorylated tau Amyloid pathology; APP, soluble Ab-peptides Cholinergic markers; glycosylated acetylcholine esterase Synaptic markers; synaptotagmin, rab3, SNAP25 Neuronal/axonal damage; Tau-protein, ASAT, 14-3-3, tissue transglutaminase Inflammatory markers; CRP, a1-antichymotrypsin, a2-macroglobulin, interleukins, complement factors Glial activation markers; GFAP, S-100b, glutamine syntethase Oxidative damage markers; 8-hydroxyguanoside, 4-hydroxynonenal SOD, isoprostanes, nitrotyrosine Others; neuronal thread protein, NO-metabolites, prostaglandins, 24S-hydroxycholesterol, heme-oxygenase 1, kallikrein

Biological Markers of Alzheimer’s Disease

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Proteins in CSF: the Amyloid Proteins Ab

and have been found in patients with other dementing diseases, such as Creutzfeld–Jakob disease (28), other neurological diseases (OND) and acute head trauma and stroke (36,37). These findings support the hypothesis that CSF tau may be a marker of neuronal cell death and axonal degeneration (36,38). Recently, four different independent bioassays have been developed which reliably detect phosphorylated tau CSF. These assays detect CSF tau phosphorylated at threonine 181 (P181), serine 199 (P199), threonine 231, serine 235, and serine 396/404; phosphorylated tau may be a more specific marker for AD than total tau (39). Since measurements of any single CSF marker alone have not proved a useful diagnostic test for AD, combinations of different markers have been used. Tau protein has been combined with neuronal thread protein (40) and with the soluble interleukin-6 receptor complex (41). These combinations have resulted in the specificity of 93% and 90%, and the sensitivity of 63% and 92%, respectively. However, these results have to be confirmed in larger patient series. Most studies have combined measurements of CSF Ab42 and tau. This combination test behaves rather well as a confirmatory test for AD, giving a specificity of approximately 90% for non-demented controls and 50–80% for other dementias (34). Our recent findings showed that the combination of Ab42 and phosphorylated tau detected patients with progressive MCI with high specificity (88%) whereas the sensitivity of the assay was low (61%) (32).

Particular interest has focused on the usefulness of proteins associated with the neuropathology of AD. Isoforms of secreted APP can be detected in blood and CSF. In addition, soluble Abs (sAbs) are secreted during normal cellular metabolism (22), and small amounts of soluble Ab-peptides are found in CSF and blood. Earlier studies concentrated on measurements of total sAb concentrations in CSF. We showed a decrease of total sAb in AD patients (23), whereas no changes were found in most studies (24). More recent studies have focused on measurements of different isoforms of sAb in CSF, particularly Ab42 due to its critical role in the early pathogenesis of AD. No differences in CSF Ab40 concentrations have been found between controls and AD patients (25,26), whereas most studies including our own have shown that Ab42 concentrations are lower in the CSF of AD patients (26,27). Lower Ab42 levels in CSF have been reported also in patients with other dementias (26), including Creutzfeldt–Jakob disease (28). Because treatment options in AD are emerging, the focus has recently been on early diagnosis. Measurements of CSF Ab42 concentrations in patients with mild cognitive impairment (MCI), a possible risk stage for AD, have shown a significant decrease in MCI patients who later developed AD (29–31). A recent population-based study showed that CSF Ab42 levels were decreased in asymptomatic subjects who developed dementia during a 3-year follow-up period (31). The odds ratio for the development of dementia was 8.2 for individuals with low CSF Ab42. Our results showed that CSF Ab42 levels were significantly decreased in MCI patients who developed AD during the follow-up whereas nonprogressive MCI patients had levels similar to controls (32). These findings suggest that CSF Ab42 may have a high predictive value for early AD.

Proteins in CSF: Tau Soluble tau protein can be detected in CSF under normal conditions (33). Several studies have shown higher levels of total CSF tau protein concentrations in AD patients compared to controls (34). Our studies suggest that elevated levels of CSF tau in AD reflect neurofibrillar pathology (35). CSF tau is increased at the early stage of AD (36), already in patients with MCI, who later develop AD (32,34). However, high CSF tau levels are not specific for AD

Plasma Amyloid Proteins Increased plasma levels of Ab42 have been reported occasionally in AD patients, particularly in patients with familial AD, in presymptomatic carriers of mutations causing familial AD (42), in first degree relatives of AD patients, and in some cognitively normal individuals who developed AD in follow-up (43). However, most sporadic AD cases do not have increased Ab42 concentrations in plasma (44). Patients with Down syndrome (DS) develop neuropathological changes consistent with AD by the time they reach 40 years of age (45) possibly due to triplication and overexpression of the APP gene. Plasma levels of Ab42 and Ab40 are increased in patients with DS (46,47), and may contribute to the development of brain amyloidosis and dementia in these subjects. We followed 30 DS patients (age 26–64 years) for 5 years (47). At baseline 21 subjects were non-

1504 demented and nine subjects were demented. The assessment of general intellectual performance and daily functioning, as well as collection of blood samples were done yearly. Eight patients developed dementia during the follow-up. There were no differences in plasma Ab40 and Ab42 levels between demented and non-demented subjects. The levels remained rather stable in individual patients during the follow-up and there was no association between the development of dementia and plasma levels of Ab40 or Ab42 during the follow-up. We conclude that plasma Ab40 and Ab42 are not useful markers for development of AD.

SEARCH FOR NEW MARKERS: EXPRESSION PROTEOMICS AND AD Unlike a genome, our proteome is extremely dynamic and constantly changing in response to any internal (e.g. aging) or external stimuli (e.g. toxic exposure). Alterations in the expression and modification of proteins may have functional consequences and lead to pathological conditions such as AD. Expression proteomics aims at the simultaneous separation and quantification of as many protein isoforms as possible, and may thus help to find new biological markers or a panel of markers useful in the more accurate diagnosis and follow-up of AD. The word proteome (=the entire PROTEin complement expressed by a genOME, or by a cell or a tissue type) was first introduced by Wilkins et al. in 1995 (48). Proteomics currently comprises a rapidly expanding network of large and small-scale techniques that are related to protein chemistry. The traditional approach in expression proteomics, two-dimensional gel electrophoresis (2-DE) (49–51) in combination with the identification of proteins by mass spectrometry and/or immunoblotting, enables the simultaneous measurement of levels of multiple protein isoforms (52). By using 2-DE, proteins are separated according to their isoelectric point (pI) in the first dimension and according to their molecular weight (Mw) in the second dimension. 2-D images are visualized and scanned followed by image analysis with specific software. It is possible to find both qualitative and quantitative differences in proteomes by comparing 2-D maps in healthy and disease states. 2-DE techniques have recently improved a great deal and a variety of applications are currently commercially available to researchers (53,54).


2-DE Studies in AD The first 2-DE studies on the levels of AD brain proteins have been carried out within the last few decades (55–57). In these studies, alterations in the levels of a number of proteins, i.e., glial fibrillary acidic protein, tubulin and phosphocreatine kinase, were already discovered. Since then, the number of 2DE studies has multiplied. Up to now, deranged levels of more than 100 brain protein isoforms have been identified in neurodegenerative disorders. Previous studies have greatly extended our knowledge on the pathogenesis of AD and may contribute to the discovery of new diagnostic markers for AD. Fig. 1 shows an example of a 2-DE map of most abundant human frontal cortex proteins in AD brain. Several 2-DE studies have been performed without successful identification of AD-specific changes in the CSF proteome (58–61). In the early 1990’s, we found that an isoform of haptoglobin-alpha)1 chains was present in the majority of 2-D maps in AD vs. age-matched controls, indicating a possible altered blood–brain-barrier function in AD (62). This was one of the first studies pointing to the possibility of screening pathological changes in the CSF of AD patients by utilizing 2-DE. Currently, there are a number of studies that have reported

Fig. 1. An example of a 2-DE map of most abundant human frontal cortex proteins. Human brain proteins were extracted as described earlier (75) and separated first according to their pI using a 18 cm long 3–10 NL Immobilized pH Gradient gel (Amersham Biosciences). In the second dimension, proteins were separated according to their Mw on a 12.5% SDS-PAGE gel. Proteins were visualized with a Sypro Ruby fluorescent stain according to the manufacturer’s instructions (Bio-Rad) and fluorescence signals were detected by fluoroimager Storm 860 (Amersham Biosciences, Uppsala, Sweden).

Biological Markers of Alzheimer’s Disease differences in the CSF proteome by comparing AD patients and controls (63,64). Despite the recent advances in 2-D CSF proteomics, it has also become apparent that only the most abundant proteins can be screened and additional fractionation steps would be needed in order to see more changes. An increasing amount of evidence also indicates that AD is in fact a systemic disorder manifesting itself in numerous abnormalities in non-neural tissues, e.g., in blood and skin cells (65,66). The first 2-DE study in which cells of non-neuronal origin were investigated suggested an abnormal expression of actin in lymphocytes (67). Thereafter, we detected variations in proteomes of red blood cell membrane, platelets and lymphocytes between AD patients and controls (68). One of the proteins identified was again the cytoskeletal protein actin that exhibited lower levels in AD platelets and lymphocytes as compared to controls. Since then, surprisingly few 2-DE studies of AD blood cell proteomes have been carried out (69). In addition to the levels of protein isoforms, posttranslational modifications such as oxidation may be studied by slightly modified 2-DE experiments. Oxidative damage of proteins has gained a lot of interest recently since oxidative stress is thought to play an important role in the pathogenesis of AD (70). Also, markers of increased oxidative stress may already be detected prior to the onset of the disease and in patients with MCI (71,72). Oxidatively modified proteins can be studied by 2-D oxyblotting. These techniques are based on the separation of proteins by 2-DE followed by the immunoblotting of carbonylated proteins with an antibody (73). By using a similar approach, we detected about 100 oxidized proteins in the frontal cortices of AD patients and age-matched controls (74). Some of the proteins were further identified by mass spectrometry and database searches. The major finding was that both cytosolic malate dehydrogenase and glutamate dehydrogenase, proteins closely related to neurotransmitter release and energy metabolism, were actually less oxidized in AD patients (75). This was surprising, since until now unaltered or more oxidized proteins have been identified in the AD brain (76) and plasma (77,78).

GENETIC MARKERS ASSOCIATED WITH AD Faults in certain genes cause AD. The first pathogenic mutations responsible for the disorder

1505 were found in the APP gene on chromosome 21 (79). Eighteen different APP mutations are known today to account for AD in 50 families of various ethnic origins (see the Alzheimer Disease and Frontotemporal Dementia Mutation Database (AD&FTDMDB), http://www.molgen.ua.ac.be/ADMutations/), reflecting the rareness of these mutations as the cause of AD. Fourteen of the mutations lead to a classical AD phenotype, whereas carriers of the remaining four APP mutations develop AD together with congophilic amyloid angiopathy/cerebral hemorrhage or hereditary cerebral hemorrhage with amyloid. A locus predisposing to AD was discovered on chromosome 14 followed by the identification of the gene, presenilin 1 (PSEN1) (80). Altogether 139 PSEN1 mutations have now been identified in 277 families (see AD&FTDMDB). 111 of these mutations result in classical AD, while in the remaining 28 cases patients also display atypical features, including spastic paraparesis, myoclonus, frontotemporal dementia, parkinsonism, epilepsy or behavioral disturbances. A third gene also involved in familial AD of early onset and mendelian inheritance was found on chromosome 1 soon after the discovery of PSEN1 and termed PSEN2 (81,82). So far, 10 different mutations have been reported in 16 pedigrees worldwide (see AD&FTDMDB). In our own study, we did not find pathological mutations in APP, PSEN1 or PSEN2 in AD (83). The causal mutations harbored by the aforementioned three genes most likely give rise to the disease via mechanisms that result in the elevated production of total Ab and/or the longer form of the Ab peptide (Ab42,43), or enhance the formation of Ab protofibrils (84). These mutations already offer a possibility to confirm the diagnosis of AD in rare familiar cases. A fourth gene contributing to susceptibility to AD is apolipoprotein E (APOE) (85). ApoE is a polymorphic glycoprotein existing in three major isoforms: apoE2, E3 and E4, which are products of three alleles: e2, e3 and e4. Among the three common variants, the e4 allele is significantly over-represented in patients afflicted with AD. The association of e4 with the disease has been confirmed in a large number of studies followed by the recognition of e4 as the most important risk factor for AD (86). APOE genotyping is not usable as a single diagnostic marker for AD but can offer an addition to the procedures utilized in diagnostics. Since some of the AD families with an autosomal dominant inheritance pattern have no mutations in the APP, PSEN1 or PSEN2 genes and, on the other

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Fig. 2. The volume of the hippocampus of an AD patient was measured from MRI (Siemens, Signa 1.5 T) pictures. The parameters were as follows: (T2 Tra T.3D 1e2mm, FOV 230 220 200, MTX 161/512 256/256 203/256, TH/Cap 3.0/0.0 3.0/0.0.2.0/0.0). The volume of the voxel is 1.47705 mm3. Results: Right hippocampus 2177 voxels 3.2 cm3, Left hippocampus 2193 voxels 3.2 cm3. Both hippocampuses are of the same size. The largest difference in the volume was 11% (slice 8).

hand, a significant proportion of sporadic AD patients do not carry APOE e4, other genetic factors contributing to the development of AD must exist (87). However, so far no new causative or susceptibility genes have been identified for AD in spite of the last 10 year’s intensive research.

IMAGING METHODS IN THE DIAGNOSIS OF AD In AD, the imaging techniques may be used in prediction and early diagnosis as well as measuring the results of treatment. The accuracy of the clinical diagnosis of AD as compared with the golden standard (autopsy findings) was 78% in a meta-analysis of 13 studies (88). The imaging methods must surpass the clinical standard and have added value in diagnostics. AD is a region-specific disorder affecting characteristic brain regions early on in the course of the disease. The most promising early imaging marker for AD is hippocampal atrophy (89,90); more generalized loss of brain volume (cerebral cortex as well as other areas) develops later and correlates with cognitive decline (91,92). Hippocampal atrophy can be found before the onset of AD (93) and progresses parallel to the clinical progressing of AD (94). Brain-computed tomography (CT) and positron emission tomography (PET) have only modest value for the positive diagnosis of AD. A recent study suggests that PET with a novel tracer, Pittsburgh Compound-B or PIB, may provide quantitative information on b-amyloid deposits in living AD patients (95). Magnetic resonance imaging (MRI) techniques, on the other hand, offer a more valuable imaging approach in AD. MRI has made it possible

to visualize the medial temporal structures (such as the hippocampus, amygdale, entorhinal cortex, parahippocampal gyrus and temporal medial lobe) that are affected early in the pathogenesis of AD (96). We have developed a semi-automatic segmentation for the volumetric analysis of the brain (97) and applied it to the segmentation of the different brain structures in AD patients. Volumetry of the hippocampal structures was performed (see Fig. 2) using a 1.5 T MRI (Siemens, Signa). The results showed that MR hippocampal atrophy has limited specificity for AD, because it is found, e.g., in hippocampal sclerosis. The very recent Consensus Statement of the Neuroimaging Work Group (NWG) of the Alzheimer’s Association (98) summarizes the current status and value of the use of MRI in diagnosis and therapeutics. In the near future, however, the improvement of functional MRI techniques, MR spectroscopy and the development of specific molecular markers for MRI will be invaluable.

CONCLUSIONS Taken together, the years spent in the search for an optimal marker for AD have been somewhat frustrating. Definitive biological markers which would help in differentiating AD from other dementing states or in monitoring the course of the illness have so far not been discovered. The biological markers currently available for AD can only be used as one tool among many in the diagnostic procedure and a physician’s evaluation of the clinical picture still is very essential for a correct diagnosis. The data on biological markers of AD gathered so far suggest that the disease is etiologically heterogeneous; therefore instead of AD the term Alzhei-

Biological Markers of Alzheimer’s Disease mer’s syndrome, as discussed earlier by Mattila and Frey (99), would depict the complexity and nature of the entity known as AD. One future approach could be the search for and confirmation of separate biological markers for certain defined subgroups of AD rather than for AD as a single entity. This could be important also for the development of more specific treatments in the future.

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