**1. Introduction**

Klivenyi P, Knudsen CS, Kummer MP, Lui J, Lladó A, Lewczuk P, Li Q-X, Martins R, Masters C, McAuliffe J, Mercken M, Moghekar A, Molinuevo JL, Montine TJ, Now‐ atzke W, O'Brien R, Otto M, Paraskevas GP, Parnetti L, Petersen RC, Prvulovic D, de Reus HPM, Rissman R a, Scarpini E, Stefani A, Soininen H, Schröder J, Shaw LM, Skinningsrud A, Skrogstad B, Spreer A, Talib L, Teunissen C, Trojanowski JQ, Tuma‐ ni H, Umek RM, Van Broeck B, Vanderstichele H, Vecsei L, Verbeek MM, Windisch M, Zhang J, Zetterberg H, Blennow K (2011) The Alzheimer's Association external quality control program for cerebrospinal fluid biomarkers. *Alzheimer's & dementia:*

[38] Oliver KG, Kettman JR, Fulton RJ. Multiplexed analysis of human cytokines by use

[39] Olsson A, Vanderstichele H, Andreasen N, De Meyer G, Wallin A, Holmberg B, et al. Simultaneous measurement of beta-amyloid(1- 42), total tau, and phosphorylated tau (Thr181) in cerebrospinal fluid by the xMAP technology. Clin Chem 2005; 51:336–45.

[40] Mattsson N, Zetterberg H, Hansson O, Andreasen N, Parnetti L, Jonsson M, et al. CSF biomarkers and incipient Alzheimer disease in patients with mild cognitive im‐

[41] Jongbloed W, Kester MI, van der Flier WM, Veerhuis R, Scheltens P, Blankenstein MA, Teunissen CE. Discriminatory and predictive capabilities of enzyme-linked im‐ munosorbent assay and multiplex platforms in a longitudinal Alzheimer's disease

[42] Schipke CG, Prokop S, Heppner FL, Heuser I, Peters O. Comparison of immunosorb‐ ent assays for the quantification of biomarkers for Alzheimer's disease in human cer‐

[43] Teunissen CE, Verwey N a, Kester MI, van Uffelen K, Blankenstein M a (2010) Stand‐ ardization of Assay Procedures for Analysis of the CSF Biomarkers Amyloid β((1-42)), Tau, and Phosphorylated Tau in Alzheimer's Disease: Report of an Interna‐ tional Workshop. International journal of Alzheimer's disease 2010, Article ID

*the journal of the Alzheimer's Association* 7, 386-395..

pairment. JAMA 2009;302:385–93.

635053, 6 pages.

192 Understanding Alzheimer's Disease

of the FlowMetrix system. Clin Chem 1998;44: 2057–60.

study. Alzheimers Dement. 2012 Oct 27. (Epub ahead of print)

ebrospinal fluid Dement Geriatr Cogn Disord. 2011;31(2):139-45.

Alzheimer's disease (AD) is a neurodegenerative disorder of the central nervous system characterized by a progressive loss of short-term memory accompanied by a gradual loss of cognitive functions (Ross et al., 2004). AD is among the most frequently encountered diseases in aging societies with an estimated 5million people in the United States and 17 million people worldwide suffering from the disease. It is expected that these numbers will quadruple by the year 2040, by which 1 out of 45 Americans will be affected, leading to a considerable public health burden (Fratiglioni et al., 1999). AD pathogenic mecha‐ nisms contributing to neuronal loss and brain dysfunction are still unclear. However, remarkable advances have taken place in understanding of both the genetics and molecular biological aspects of the intracellular processing of amyloid and tau and the changes leading to the pathologic formation of extracellular amyloid plaques and the intraneuronal aggregation of hyperphosphorylated tau into neurofibrillary tangles. This progress in our understanding of the molecular pathology has set the stage for clinical‐ ly meaningful advances in the development of biomarkers.

Proper diagnosis is essential for instituting appropriate clinical management. While diagnostic accuracy for the disease has improved, the differential diagnosis of the disorder is still problematic. In the very early stages of the disease, frequently classified as mild cognitive impairment (MCI), delineating disease process from "normal ageing" may be difficult; in later stages of the disease, distinguishing AD from a number of neurodegener‐ ative diseases associated with dementia may also be difficult. Furthermore, the disease progression is slow and there is variability of performance on clinical measures, making it difficult to monitor change effectively. Since disease modifying therapy is likely to be most effective early in the course of disease, early diagnosis is highly desirable before neurodegeneration becomes severe and widespread.

© 2013 Binukumar and Pant; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

In clinical practice, the diagnosis of AD is still largely based on consensuscriteria combined with the exclusion of secondary causes of memory loss (Knopman et al., 2001; McKhann et al., 1984).Thus, there is an urgent and desperate need for a biomarker that can reliably prognose the disease. Biomarkers of AD occupy an essential place in recently formulated diagnostic criteria for AD, in which their role is to identify the pathophysio‐ logical processes underlying cognitive impairment or to help predict time to reach up to dementia. Criteria for a useful biomarker have been proposed by an international consensus group on molecular and biochemical markers of AD in 1998 (The Ronald and Nancy Reagan Research Institute of the Alzheimer's Association and the National Institute on Aging Working Group, 1998). According to these guidelines, a biomarker for AD should detect a manifestation of the fundamental neuropathology and be validated in neuropathologically-confirmed cases. Its sensitivity for detecting AD should exceed 80% and its specificity in differentiating between AD and other dementias should be higher than 80%. Ideally, a biomarker should also be reliable, reproducible, non-invasive, simple to perform, and inexpensive. One further role of particular interest to patients and clinicians dealing with AD is its ability to detect the disease at the earliest possible stage.

**Category Reference Sensitivity range**

Tau Arai et al.,

Tau Riemenschnei

Tau Shoiji et al., 1998

Tau Kanai et al., 1998

Tau Tapiola et al., 1998

Tau Kahle et al., 2000

Tau Sjögren et al., 2000

Tau Shoji et al., 2002

Tau Buerger et al., 2002

Tau Riemenschnei

Tau Schönknecht

der et al., 2002

et al., 2003

Data from Blennow K, Hampel H (2003)

**Table 1.** CSF total tau (T-tau) as a diagnostic marker for AD

1995

der et al., 1996

**(100%) for AD versus controls** **Methods Study Title Study population**

AD (n=70), non-AD (n=96) control (n=19)

Candidate Bio-Markers of Alzheimer's Disease

http://dx.doi.org/10.5772/55069

195

AD(n=22), dementia(n=3) Healthy controls(HC)(n=19)

sporadic AD(n=55), controls(n=34), non-AD dementia(n=23), other neurological diseases(n=45)

AD(n=93), non-AD dementia(n=33) other neurological diseases (n=56), HC(n=54)

Early AD(n=81), other dementia (n=43), non demented neurologic HC(n=33)

Probable AD(n=25), definite AD(n=5), non demented with PD (n=29), HC(n=16).

AD (n = 19), FTD (n = 14), ALS (n = 11) PD( n = 15)

AD(n=366), 168 non-AD dementia(n=168) HC(n=181).

AD(n=82) FTD(n=26) VD(n=20) HC(n=21)

FTD(n=34), AD(n=74), HC(n=40).

manifest AD (n=43) Incipient AD(n=8) VD(n=16)

HC(n=16)

HC(n = 17)

diagnostic marker in Alzheimer's

early Alzheimer's disease.

1-40 and A beta 1-42(43) as a biochemical marker of Alzheimer's

fluid levels of tau, A beta1-40, and A beta1-42(43) in Alzheimer's disease: a

genotype in early Alzheimer's disease.

neuronal thread protein in Alzheimer's

Alzheimer's disease and amyotrophic lateral sclerosis may reflect mismetabolism of -amyloid induced by

disorders:a large scale multicenter study

disease with cerebrospinal fluid levels of tau protein phosphorylated at

80-90 ELISA Tau in cerebrospinal fluid: a potential

90-100 ELISA Cerebrospinal protein tau is elevated in

20-30 ELISA Combination assay of CSF tau, A beta

30-40 ELISA Longitudinal study of cerebrospinal

50-60 ELISA CSF tau is related to apolipoprotein E

50-60 ELISA Combined assessment of tau and

60-70 ELISA Decreased CSF -amyloid42 in

50-60 ELISA Cerebrospinal fluid tau in dementia

70-80 ELISA Differential diagnosis of Alzheimer's

80-90 ELISA Tau and Abeta42 protein in CSF of

50-60 ELISA Levels of total tau and tau protein

disease

disease

study in Japan

disease CSF

separate mechanisms

by a Japanese study group

patients with frontotemporal

phosphorylated at threonine 181 in patients with incipient and manifest

threonine 231

degeneration

Alzheimer's disease

Based on growing body of evidence concerning the pathophysiology of AD, a number of putative biological markers of disease have been evaluated against clinical and neuropa‐ thological standards. Biomarkers are very useful for diagnosing and monitoring disease progression (Ward et al., 2007) and are important for patient selection, monitoring sideeffects, aiding selection of appropriate patient treatment, and helping new drug discov‐ ery. For the clinical studies of AD therapeutics, there is an increasing need for diagnostic markers to ensure that therapies are targeted at the right patient population, to initiate early treatment when disease-modifying drugs will be available, and to monitor disease progression (Hye et al., 2006).

#### **2. Biomarkers in CSF**

One of the most promising sources of biomarkers in AD is the cerebrospinal fluid (CSF).The molecular changes in the brain extracellular and interstitial environments are reflected in CSF. The single-cell layer epithelium separating the two compartments allows a virtually unhindered flow of molecules from the brain towards the CSF. CSF biomark‐ ers for AD should reflect the central pathogenic processes in the brain. Furthermore the CSF is accessible to trained clinicians using a relatively simple lumbar puncture (Fenton et al., 1994). Several studies have investigated CSF inflammatory markers, immunologi‐ cal mediators, neurotrophins, metalloproteinases or isoprostenes. Candidate CSF biomark‐ ers include total tau (T-tau) as a marker for the neuronal degeneration (table 1), phosphorylated tau (P-tau) as a marker for tau hyperphosphorylation (table 2) and formation of tangles A*β*42 as a marker for A*β* metabolism and plaque formation (table 3, Blennow et al.,2003).


Data from Blennow K, Hampel H (2003)

In clinical practice, the diagnosis of AD is still largely based on consensuscriteria combined with the exclusion of secondary causes of memory loss (Knopman et al., 2001; McKhann et al., 1984).Thus, there is an urgent and desperate need for a biomarker that can reliably prognose the disease. Biomarkers of AD occupy an essential place in recently formulated diagnostic criteria for AD, in which their role is to identify the pathophysio‐ logical processes underlying cognitive impairment or to help predict time to reach up to dementia. Criteria for a useful biomarker have been proposed by an international consensus group on molecular and biochemical markers of AD in 1998 (The Ronald and Nancy Reagan Research Institute of the Alzheimer's Association and the National Institute on Aging Working Group, 1998). According to these guidelines, a biomarker for AD should detect a manifestation of the fundamental neuropathology and be validated in neuropathologically-confirmed cases. Its sensitivity for detecting AD should exceed 80% and its specificity in differentiating between AD and other dementias should be higher than 80%. Ideally, a biomarker should also be reliable, reproducible, non-invasive, simple to perform, and inexpensive. One further role of particular interest to patients and clinicians dealing with AD is its ability to detect the disease at the earliest possible stage.

Based on growing body of evidence concerning the pathophysiology of AD, a number of putative biological markers of disease have been evaluated against clinical and neuropa‐ thological standards. Biomarkers are very useful for diagnosing and monitoring disease progression (Ward et al., 2007) and are important for patient selection, monitoring sideeffects, aiding selection of appropriate patient treatment, and helping new drug discov‐ ery. For the clinical studies of AD therapeutics, there is an increasing need for diagnostic markers to ensure that therapies are targeted at the right patient population, to initiate early treatment when disease-modifying drugs will be available, and to monitor disease

One of the most promising sources of biomarkers in AD is the cerebrospinal fluid (CSF).The molecular changes in the brain extracellular and interstitial environments are reflected in CSF. The single-cell layer epithelium separating the two compartments allows a virtually unhindered flow of molecules from the brain towards the CSF. CSF biomark‐ ers for AD should reflect the central pathogenic processes in the brain. Furthermore the CSF is accessible to trained clinicians using a relatively simple lumbar puncture (Fenton et al., 1994). Several studies have investigated CSF inflammatory markers, immunologi‐ cal mediators, neurotrophins, metalloproteinases or isoprostenes. Candidate CSF biomark‐ ers include total tau (T-tau) as a marker for the neuronal degeneration (table 1), phosphorylated tau (P-tau) as a marker for tau hyperphosphorylation (table 2) and formation of tangles A*β*42 as a marker for A*β* metabolism and plaque formation (table 3,

progression (Hye et al., 2006).

**2. Biomarkers in CSF**

194 Understanding Alzheimer's Disease

Blennow et al.,2003).

**Table 1.** CSF total tau (T-tau) as a diagnostic marker for AD


**Catagory Reference Sensitivity**

Aβ1-42 Galasko et al ., 1998

Aβ1-42 Andreasen et al., 1999

Aβ1-42 Andreasen et al.,1999

Aβ1-42 Andreasen et al., 1999C

Aβ1-42 Hulstaert et al., 1999

Aβ1-42 Otto et al., 2000

Aβ1-42 Kapaki et al., 2001

Aβ1-42 Sjögren et al., 2002

Data from Blennow K, Hampel H.(2003)

**Table 3.** CSF Aβ1-42as a diagnostic marker for AD

**(100%) for AD versus controls**

**Methods Study Title Study group**

levels in the clinical diagnosis of Alzheimer disease and relation to apolipoprotein E genotype

disease: differences between early- and late-onset Alzheimer disease and stability during the course of

disease: differences between early- and late-onset Alzheimer disease and stability during the course of Probable AD(n=82), control (n=60) ND (n= 74)

Candidate Bio-Markers of Alzheimer's Disease

http://dx.doi.org/10.5772/55069

197

AD (n=53) Control (n=21)

AD (n= 407) Depression(n=28) control (n=65).

AD (n=53) Control (n= 21)

AD (n=150) control (n= 100) ND (n=84),

CJD (n=27), AD(n=14), other dementia(n=19), NDC(n=20)

CJD (n=14), AD(n=38)controls

AD (n = 19), FTD (n = 14), ALS (n = 11) PD ( n = 15) controls (n = 17).

(n=47).

70-80 ELISA High cerebrospinal fluid tau and low amyloid beta42

90-100 ELISA Cerebrospinal fluid -amyloid(1-42) in Alzheimer's

90-100 ELISA Sensitivity, specificity and stability of CSF t-tau in AD

80-90 ELISA Cerebrospinal fluid -amyloid(1-42) in Alzheimer's

70-80 ELISA Improved discrimination of AD patients using beta-

90-100 ELISA Decreased beta-amyloid1-42 in cerebrospinal fluid

70-80 ELISA Highly increased CSF tau protein and decreased

90-100 ELISA Decreased CSF -amyloid42 in Alzheimer's disease

mechanisms

from Alzheimer's disease?

amyloid(1-42) and tau levels in CSF.

of patients with Creutzfeldt-Jakob disease

beta-amyloid (1-42) in sporadic CJD: a discrimination

and amyotrophic lateral sclerosis may reflect mismetabolism of -amyloid induced by separate

in a community-based patient sample.

disease

disease.

**Table 2.** CSF Phosphorylaterd tau (p-tau) as a diagnostic marker for AD


**Catagory Reference Sensitivity**

p- tau Ishiguro etal., 1999

196 Understanding Alzheimer's Disease

p- tau Kohnken et al., 2000

p- tau Sjögren et al., 2001

p- tau Parnetti et al., 2001

p-tau Sjögren et al., 2002

p- tau Buerger et al., 2002

p-tau Schönknecht et al.,2003

Data from Blennow K, Hampel H.(2003)

**(100%) for AD versus controls**

p- tau Itoh et al.,2001 90-100 ELISA Large-scale, multicenter study of

p-tau Hu et al., 2002 90-100 ELISA Levels of nonphosphorylated and

**Table 2.** CSF Phosphorylaterd tau (p-tau) as a diagnostic marker for AD

**Methods Study Title Study population**

AD (n=36) , Controls

AD(n=27), non-AD(n=31)

FTD( n = 14), AD( n = 47) VAD( n = 16), controls (n = 12)

AD( n = 236), non-AD (n = 239), controls (n =

AD (n=80), DLB (n=43) Controls (n=40)

AD (n = 19), FTD (n = 14), ALS (n = 11) PD( n

MCI(n=77), probable AD (n=55) Control

AD (*n* = 30), VaD, (*n* = 18) non-AD (*n* = 13): depression (*n* = 3), malignant lymphoma (*n* = 2) control (*n* = 24)

AD (n=80) DLB (n=43) Controls (n=40).

95)

= 15)

(n=30)

(n=30)

fluid is a diagnostic marker for Alzheimer's

threonine 231 in cerebrospinal fluid of Alzheimer's disease patients

associated protein-43 and soluble amyloid precursor protein correlate in Alzheimer's

for discriminating AD from dementiawith Lewy bodies. Phospho-Tau International

disease and amyotrophic lateral sclerosis may reflect mismetabolism of -amyloid induced by separate mechanisms.

231 correlates with cognitive decline in MCI

phosphorylated tau in cerebrospinal fluid of

for discriminating Alzheimer's disease from dementia with Lewy bodies. Phospho-Tau

Alzheimer's disease patients: an ultrasensitive bienzyme-substrate-recycle enzyme-linked immunosorbent assay.

International Study Group

disease, reflecting a common pathophysiological process

cerebrospinal fluid tau protein phosphorylated at serine 199 for the antemortem diagnosis of AD

80-90 ELISA Phosphorylated tau in human cerebrospinal

40-50 ELISA The cerebrospinal fluid levels of tau, growth-

80-90 ELISA CSF phosphorylated tau is a possible marker

Study Group

90-100 ELISA CSF tau protein phosphorylated at threonine

subjects

60-70 ELISA CSF phosphorylated tau is a possible marker

50-60 ELISA Decreased CSF -amyloid42 in Alzheimer's

disease.

80-90 ELISA Detection of tau phosphorylated at

**Table 3.** CSF Aβ1-42as a diagnostic marker for AD

#### **2.1. Tau protein**

One of the major neuropathological hallmarks of AD are neurofibrillary tangles composed of paired helical filaments (PHF). The principal protein subunit of PHF is abnormally phos‐ phorylated tau (p-tau) (Iqbal et al., 1998). Physiologically, tau protein is located in neuronal axons, in components of the cytoskeleton and in the intracellular transport systems. Total-tau (t-tau) and truncated forms of monomeric and p-tau can be traced in the CSF. Using antibodies that detect all isoforms of tau proteins independent of phosphorylation, or specific phosphor‐ ylation Core biomarker candidates of Alzheimer's disease 251 sites, ELISA have been devel‐ oped to measure t-tau and p-tau concentrations (Vandermeeren et al., 1993; Blennow et al., 2002, 1995; Hampel et al., 2003). CSF total tau protein in the differentiation between AD and normal aging. Total tau protein, thought to be a general marker of neuronal destruction, has been intensely studied in more than 2200 AD patients and 1000 age-matched elderly controls over the last 10 years (Sunderland et al., 2003, table 1). The most consistent finding is a statistically significant increase of CSF t-tau protein in AD. The mean level of CSF t-tau protein concentration is about 3 times higher in AD compared to elderly controls. A sensitivity and specificity level varies between studies primarily due to the different control groups used. Specificity levels between 65% and 86% and sensitivity levels between 40% and 86% have been found (Blennow et al., 2001, table 1). In several studies, a significant elevation was also found in patients with early dementia (Galasko et al., 1997; Kurz et al., 1998; Riemenschneider et al., 1997). In these studies of early dementia, the potential of CSF t-tau protein to discriminate between AD and normal aging appeared high, with average 75% sensitivity and 85% specif‐ icity. An age-associated increase of t-tau protein has been shown in nondemented subjects (Buerger et al., 2003; Sjogren et al., 2001b). Therefore, the effect of age should be considered when t-tau protein levels are employed diagnostically.

Jacob disease, and p-tau are increased by three fold in the CSF of confirmed AD patients (Shaw et al.,2009). Of the 40 or so phosphorylation sites on tau, pThr181 (phosphothreonine-181), pSer199, pSer202/pThr205 (AT8, epitopes site), pSer214/pSer212 (AT100, epitopessite), pThr231/ pSer235 (TG3 site) and pSer396/pSer396 (PHF1 site)–have been associated with tau hyperphosphorylation and to screen NCEs for potential ''tau kinase'' inhibitory activity. While pSer199 and pThr231 (p-tau231) have been evaluated as CSF biomarkers (Buerger et al., 2002; Engelborghs et al., 2008., table 2), pThr181 (also designated as p-tau181 or P-Tau181P) is the most widely used CSF biomarker to assess tau hyperphosphorylation (Lewczuk et al., 2002; Hampel et al., 2004) having similar diagnostic accuracy to p-tau231 (Fagan et al., 2009, table 2). Like Ab42, the diagnostic value of both t-tau and p-tau181 has been questioned in terms of

Candidate Bio-Markers of Alzheimer's Disease

http://dx.doi.org/10.5772/55069

199

Extracellular senile plaques consisting of beta-amyloid-protein (Aβ) are one of the histopa‐ thological hallmarks of AD (Hyman and Trojanowski., 1997). They are the source of a patho‐ genic protein with 42 amino acids (Aβ1–42) (Selkoe et al., 1993). Several groups have developed and studied different bioassays specifically designed for Ab1–42 protein (Arai et al., 1997c, Sunderland et al., 2003). The reduction in CSF Ab1–42 found in AD has been hypothesized to indirectly reflect the amyloid deposition in senile plaques (SP), resulting in lower CSF levels in AD. A marked reduction in CSF Ab1–42, however, is also found in CJD, even in cases without

To date, at least 900 patients with clinical AD and 500 healthy individuals have been enrolled in independent research studies (Andreasen et al., 2001; Andreasen et al., 1999; Galasko et al., 1998; Sunderland et al., 2003., table 3). The most consistent finding is a marked de‐ crease in Aβ1–42 protein in AD (to approximately 50% of control levels). Using Ab1–42 protein alone yielded sensitivities varying from 78% to 100% (table 3) and specificities from 47% to 81% when distinguish AD from elderly controls. There is a pronounced overlap, however, between studiesfrom different groups. Based on recent data a cut-off-level of >500 pg=ml has been suggested to discriminate AD best from normal aging (Sjogren et al., 2001a). One study has documented a significant decrease in CSF Aβ1–42 protein in MCI subjects compared to controls, but this study had no follow-up measure (Andreasen et al., 1999a). A second study examined MCI patients who went on to develop AD. However, in this sample Aβ1–42 protein levels did not differ significantly from age-matched normal controls (Maruyama et al., 2001). Blennow et al (2003) found Ab1–42 protein to be an indicator of early identification of AD in MCI subjects taking potential confounding factors into account such as age, severity of cognitive decline, time of observation, apolipoprotein E epsilon (e) 4 (APOE e4) carrier status, and gender (Blennow et al., 2003).Studies correlating CSF Aβ1– 42 protein concentrations with cognitive performance in AD have been contradictory. Crosssectionally, the concentration of Aβ1–42 protein and cognitive measures were either inversely correlated (Kanai et al., 1998; Samuels et al., 1999) or no significant correlation was found (Andreasenet al., 1999b; Hulstaert et al., 1999; Okamura et al., 1999). In a rare longitudinal study, a decrease in CSF Aβ1–42 protein was documented overa three year

their specificity as AD biomarkers (Mattsson et al., 2009).

Ab-positive plaques (Kapaki et al., 2001; Otto et al., 2000., table 3).

**2.3. β-Amyloid-protein**

#### **2.2. Phosphorylated tau (p-tau)**

Tau protein exists in six isoforms of 352–441 amino acids in length that are subject to a variety of posttranslational modifications (Hanger et al., 2007) and, presumably, function. Of the 79 serine and threonine phosphorylation sites on the longest isoform of tau, 4R/2N, approxi‐ mately 40 have been verified (Iqbal et al., 2010) of which 25 have been identified as sites of ''abnormal phosphorylation'' (Mazanetz et al., 2007). The phosphorylation state of tau is the net result of a balance of kinase and phosphatase activity. Much of the activity in tau-based drug discovery has been focused on selective finding inhibitors of ''tau kinase'', a combination of the activity of two serine/theronine kinases that can phosphorylate tau – glycogen synthase kinase 3 (GSK3; tau protein kinase I), cyclin-dependent kinase 5 (CDK5; tau protein kinase II) and a third kinase, extracellular signal-regulated kinase 2 (ERK2), from the possible 518 member kinase family, as a possible therapeutic approach to treating AD (Hanger et al.,2009 Mazanetz et al.,2007, Brunden et al., 2009). Other kinases that are possible targets to prevent tau hyperphosphorylation are casein kinase 1 (Hanger et al.,2007), AMP-activated protein kinase (AMPK) (Greco et al.,2009) and DYRK1A and AKAP-13 (Azorsa et al., 2010). From a biomarker perspective, t-tau, a generic measure of cortical axon damage associated with AD, multiple sclerosis (Hernandez et al., 2007, Bartosik-Psujek et al.,2006), stroke and CreuzfeldtJacob disease, and p-tau are increased by three fold in the CSF of confirmed AD patients (Shaw et al.,2009). Of the 40 or so phosphorylation sites on tau, pThr181 (phosphothreonine-181), pSer199, pSer202/pThr205 (AT8, epitopes site), pSer214/pSer212 (AT100, epitopessite), pThr231/ pSer235 (TG3 site) and pSer396/pSer396 (PHF1 site)–have been associated with tau hyperphosphorylation and to screen NCEs for potential ''tau kinase'' inhibitory activity. While pSer199 and pThr231 (p-tau231) have been evaluated as CSF biomarkers (Buerger et al., 2002; Engelborghs et al., 2008., table 2), pThr181 (also designated as p-tau181 or P-Tau181P) is the most widely used CSF biomarker to assess tau hyperphosphorylation (Lewczuk et al., 2002; Hampel et al., 2004) having similar diagnostic accuracy to p-tau231 (Fagan et al., 2009, table 2). Like Ab42, the diagnostic value of both t-tau and p-tau181 has been questioned in terms of their specificity as AD biomarkers (Mattsson et al., 2009).

#### **2.3. β-Amyloid-protein**

**2.1. Tau protein**

198 Understanding Alzheimer's Disease

One of the major neuropathological hallmarks of AD are neurofibrillary tangles composed of paired helical filaments (PHF). The principal protein subunit of PHF is abnormally phos‐ phorylated tau (p-tau) (Iqbal et al., 1998). Physiologically, tau protein is located in neuronal axons, in components of the cytoskeleton and in the intracellular transport systems. Total-tau (t-tau) and truncated forms of monomeric and p-tau can be traced in the CSF. Using antibodies that detect all isoforms of tau proteins independent of phosphorylation, or specific phosphor‐ ylation Core biomarker candidates of Alzheimer's disease 251 sites, ELISA have been devel‐ oped to measure t-tau and p-tau concentrations (Vandermeeren et al., 1993; Blennow et al., 2002, 1995; Hampel et al., 2003). CSF total tau protein in the differentiation between AD and normal aging. Total tau protein, thought to be a general marker of neuronal destruction, has been intensely studied in more than 2200 AD patients and 1000 age-matched elderly controls over the last 10 years (Sunderland et al., 2003, table 1). The most consistent finding is a statistically significant increase of CSF t-tau protein in AD. The mean level of CSF t-tau protein concentration is about 3 times higher in AD compared to elderly controls. A sensitivity and specificity level varies between studies primarily due to the different control groups used. Specificity levels between 65% and 86% and sensitivity levels between 40% and 86% have been found (Blennow et al., 2001, table 1). In several studies, a significant elevation was also found in patients with early dementia (Galasko et al., 1997; Kurz et al., 1998; Riemenschneider et al., 1997). In these studies of early dementia, the potential of CSF t-tau protein to discriminate between AD and normal aging appeared high, with average 75% sensitivity and 85% specif‐ icity. An age-associated increase of t-tau protein has been shown in nondemented subjects (Buerger et al., 2003; Sjogren et al., 2001b). Therefore, the effect of age should be considered

Tau protein exists in six isoforms of 352–441 amino acids in length that are subject to a variety of posttranslational modifications (Hanger et al., 2007) and, presumably, function. Of the 79 serine and threonine phosphorylation sites on the longest isoform of tau, 4R/2N, approxi‐ mately 40 have been verified (Iqbal et al., 2010) of which 25 have been identified as sites of ''abnormal phosphorylation'' (Mazanetz et al., 2007). The phosphorylation state of tau is the net result of a balance of kinase and phosphatase activity. Much of the activity in tau-based drug discovery has been focused on selective finding inhibitors of ''tau kinase'', a combination of the activity of two serine/theronine kinases that can phosphorylate tau – glycogen synthase kinase 3 (GSK3; tau protein kinase I), cyclin-dependent kinase 5 (CDK5; tau protein kinase II) and a third kinase, extracellular signal-regulated kinase 2 (ERK2), from the possible 518 member kinase family, as a possible therapeutic approach to treating AD (Hanger et al.,2009 Mazanetz et al.,2007, Brunden et al., 2009). Other kinases that are possible targets to prevent tau hyperphosphorylation are casein kinase 1 (Hanger et al.,2007), AMP-activated protein kinase (AMPK) (Greco et al.,2009) and DYRK1A and AKAP-13 (Azorsa et al., 2010). From a biomarker perspective, t-tau, a generic measure of cortical axon damage associated with AD, multiple sclerosis (Hernandez et al., 2007, Bartosik-Psujek et al.,2006), stroke and Creuzfeldt-

when t-tau protein levels are employed diagnostically.

**2.2. Phosphorylated tau (p-tau)**

Extracellular senile plaques consisting of beta-amyloid-protein (Aβ) are one of the histopa‐ thological hallmarks of AD (Hyman and Trojanowski., 1997). They are the source of a patho‐ genic protein with 42 amino acids (Aβ1–42) (Selkoe et al., 1993). Several groups have developed and studied different bioassays specifically designed for Ab1–42 protein (Arai et al., 1997c, Sunderland et al., 2003). The reduction in CSF Ab1–42 found in AD has been hypothesized to indirectly reflect the amyloid deposition in senile plaques (SP), resulting in lower CSF levels in AD. A marked reduction in CSF Ab1–42, however, is also found in CJD, even in cases without Ab-positive plaques (Kapaki et al., 2001; Otto et al., 2000., table 3).

To date, at least 900 patients with clinical AD and 500 healthy individuals have been enrolled in independent research studies (Andreasen et al., 2001; Andreasen et al., 1999; Galasko et al., 1998; Sunderland et al., 2003., table 3). The most consistent finding is a marked de‐ crease in Aβ1–42 protein in AD (to approximately 50% of control levels). Using Ab1–42 protein alone yielded sensitivities varying from 78% to 100% (table 3) and specificities from 47% to 81% when distinguish AD from elderly controls. There is a pronounced overlap, however, between studiesfrom different groups. Based on recent data a cut-off-level of >500 pg=ml has been suggested to discriminate AD best from normal aging (Sjogren et al., 2001a). One study has documented a significant decrease in CSF Aβ1–42 protein in MCI subjects compared to controls, but this study had no follow-up measure (Andreasen et al., 1999a). A second study examined MCI patients who went on to develop AD. However, in this sample Aβ1–42 protein levels did not differ significantly from age-matched normal controls (Maruyama et al., 2001). Blennow et al (2003) found Ab1–42 protein to be an indicator of early identification of AD in MCI subjects taking potential confounding factors into account such as age, severity of cognitive decline, time of observation, apolipoprotein E epsilon (e) 4 (APOE e4) carrier status, and gender (Blennow et al., 2003).Studies correlating CSF Aβ1– 42 protein concentrations with cognitive performance in AD have been contradictory. Crosssectionally, the concentration of Aβ1–42 protein and cognitive measures were either inversely correlated (Kanai et al., 1998; Samuels et al., 1999) or no significant correlation was found (Andreasenet al., 1999b; Hulstaert et al., 1999; Okamura et al., 1999). In a rare longitudinal study, a decrease in CSF Aβ1–42 protein was documented overa three year follow-up period (Tapiola et al., 2000). A highly significant correlation between low CSF concentrations at baseline and follow up. In a separate study, no correlation was found between CSF levels and duration or severity of AD (Andreasen et al., 1999b).

In addition to hyperphosphorylated- tau, recently we have demonstrated the direct evidence of aberrantly and hyperphosphorylated neuronal intermediated proteins (NF-M/H) as integral part of NFTs of AD brain using phosphoproteomics (Rudrabhatla et al., 2011., table 5). Although, NFs have been shown immunohistologically to be part of NFTs, there has been debate that the identity of NF proteins in NFTs is due to the cross-reactivity of phosphorylated NF antibodies with phospho-Tau. This study has provided a direct evidence on the identity of NFs in NFTs by immunochemical and mass spectrometric analysis. For these studies purified NFTs were used and liquid chromatography/tandem mass spectrometry of NFT tryptic digests were analysed (table 4-6). The phosphoproteomics of NFTs clearly identified NF-M phosphopeptides (table 5). Western blotting of purified tangles with SMI31 showed a 150-kDa band corresponding to phospho-NF-M, while RT97 antibodies detected phospho-NF-H. These observations suggest that expression of some of these genes is elevated in AD in addition to their phosphorylation. Apart from phosphor Tau, phosphopeptides corresponding to MAP1B to Ser1270, Ser1274, and Ser1779); and MAP2 (corresponding to Thr350, Ser1702, and Ser1706) were also identified (table 6). These studies independently demonstrate that NF and other microtubule proteins are part of NFTs in AD brains (Rudrabhatla et al., 2011). These promising findings call for further studies on the diagnostic potential of specific antibodies derived from aberrantly and hyperphosphorylated neuronal intermediate filament (NF-M/H)

Candidate Bio-Markers of Alzheimer's Disease

http://dx.doi.org/10.5772/55069

201

peptides from AD brain as bio markers for early AD detection

**Phosphopeptide Phosphorylation site**

TPPAPKT\*PPSSGEPPK Thr181 TPPAPKTPPS\*SGEPPK Ser184 TPPAPKTPPSS\*GEPPK Ser185

TPSLPT\*PPTR Thr217 TDHGAEIVYKS\*PVVSGDTSPR Ser396 TDHGAEIVYKSPVVS\*GDTSPR Ser400

VAVVRT\*PPKS\*PSSAK Thr231, Ser235 SRT\*PSLPT\*PPTR Thr212, Thr217

TDHGAEIVYKS\*PVVSGDT\*SPR Ser396, Thr403

**Table 4.** Phosphopeptides and phosphorylation sites identified in NFT Tau

**Phosphopeptides Phosphorylation sites**

**Table 5.** Phosphopeptides and phosphorylation sites identified in NF-M and NF-H

NF-M SPVPKS\*PVEEAK Ser685 NF-M KAES\*PVKEEAVAEVVTITK Ser736 NF-M VSGSPSS\*GFRSQSWSR Ser33 NF-H EPDDAKAKEPS\*K Ser942

#### **2.4. Combination of CSF amyloid and tau phosphorylation**

The current limitations of the predictive value of Aβ 42, t-tau and p-tau181 as AD biomarkers alone, these have been used together to develop a ''CSF AD signature'', again, with mixed results (Shaw et al., 2009;Mattsso et al., 2009;Kauwe et al., 2009;Mihaescu et al., 2010;Breno et al.,2008;De Meyer et al., 2010). While some studies indicate that the combination Aβ 42, t-tau and p-tau181 biomarker signature in CSF has high predictivity in identifying cases of prodro‐ mal AD in MCI patents (Shaw et al.,2009; Jack et al.,2010; Hansson et al., 2006), there is considerable intersite variability that can confound biomarker accuracy (Kauwe et al., 2009). Reduced CSF Aβ 42 and increased CSF p-tau181 concentrations – were used independently of a clinical diagnosis to stratify patient groups (De Meyer et al., 2010). This AD signature was found in 90%, 72%, and 36% of patients with AD, mild MCI, and cognitively normal groups respectively (De Meyer et al., 2010). The cognitively normal group with an AD signature were enriched in apolipoprotein E4 alleles. Validation of these findings in two further data sets showed that 64/68 (94% sensitivity) of autopsy-confirmed AD patients were classified with an AD signature while 57 MCI patients followed for 5 years had a sensitivity of 100% in pro‐ gressing to AD based on their biomarker signature. The presence of a CSF AD signature in cognitively normal subjects was interpreted by the authors as an indication of AD pathology being present and detectable far earlier than previously envisioned in disease progression.

#### **2.5. NF proteins**

Neurofilaments (NFs) are neuron-specific intermediate filaments and serve as a major cytos‐ keletal component in neurons. In a mature mammalian neuron, NFs are co-assembled from three subunits, termed NF-H (high), NF-M (medium) and NF-L (low). As NFs are confined to the nervous system, they might be one of the best markers reflecting neuronal pathogenic changes seen in some neurological disorders, such as AD. In AD brain, the levels of phos‐ phorylated NF-H/M (pNF-H/M) have been found to be markedly increased (Wang et al., 2001). Hu et al., (2002) found that, the levels of phosphorylated NF-H/M (pNF-H/M), nonphosphorylated NF-H/M (npNF-H/M) and NF-L were significantly higher (pNF-H/M,,12–24 fold; npNF-H/M,,3–4-fold) in neurologically healthy aged people than young individuals. In AD, the levels of npNF-H/M, and NF-L were similar to vascular dementia (VaD), and higher than in age-matched controls and the levels of pNF-H/M were significantly higher AD and ALS than in aged controls and VaD. Based on these findings, it is suggested that the increased level of total NF, p-NF proteins in CSF could be used as a marker for brain aging and neuro‐ degenerative disorders in general, and the levels of pNF-H/M as a marker to discriminate AD from normal brain aging and as well as neurological conditions including VaD (Hu et al 2002).

Specific antibodies derived from aberrantly and hyperphosphorylated neuronal intermediate filament peptides from AD brain as bio markers for early AD detection

In addition to hyperphosphorylated- tau, recently we have demonstrated the direct evidence of aberrantly and hyperphosphorylated neuronal intermediated proteins (NF-M/H) as integral part of NFTs of AD brain using phosphoproteomics (Rudrabhatla et al., 2011., table 5). Although, NFs have been shown immunohistologically to be part of NFTs, there has been debate that the identity of NF proteins in NFTs is due to the cross-reactivity of phosphorylated NF antibodies with phospho-Tau. This study has provided a direct evidence on the identity of NFs in NFTs by immunochemical and mass spectrometric analysis. For these studies purified NFTs were used and liquid chromatography/tandem mass spectrometry of NFT tryptic digests were analysed (table 4-6). The phosphoproteomics of NFTs clearly identified NF-M phosphopeptides (table 5). Western blotting of purified tangles with SMI31 showed a 150-kDa band corresponding to phospho-NF-M, while RT97 antibodies detected phospho-NF-H. These observations suggest that expression of some of these genes is elevated in AD in addition to their phosphorylation. Apart from phosphor Tau, phosphopeptides corresponding to MAP1B to Ser1270, Ser1274, and Ser1779); and MAP2 (corresponding to Thr350, Ser1702, and Ser1706) were also identified (table 6). These studies independently demonstrate that NF and other microtubule proteins are part of NFTs in AD brains (Rudrabhatla et al., 2011). These promising findings call for further studies on the diagnostic potential of specific antibodies derived from aberrantly and hyperphosphorylated neuronal intermediate filament (NF-M/H) peptides from AD brain as bio markers for early AD detection


follow-up period (Tapiola et al., 2000). A highly significant correlation between low CSF concentrations at baseline and follow up. In a separate study, no correlation was found

The current limitations of the predictive value of Aβ 42, t-tau and p-tau181 as AD biomarkers alone, these have been used together to develop a ''CSF AD signature'', again, with mixed results (Shaw et al., 2009;Mattsso et al., 2009;Kauwe et al., 2009;Mihaescu et al., 2010;Breno et al.,2008;De Meyer et al., 2010). While some studies indicate that the combination Aβ 42, t-tau and p-tau181 biomarker signature in CSF has high predictivity in identifying cases of prodro‐ mal AD in MCI patents (Shaw et al.,2009; Jack et al.,2010; Hansson et al., 2006), there is considerable intersite variability that can confound biomarker accuracy (Kauwe et al., 2009). Reduced CSF Aβ 42 and increased CSF p-tau181 concentrations – were used independently of a clinical diagnosis to stratify patient groups (De Meyer et al., 2010). This AD signature was found in 90%, 72%, and 36% of patients with AD, mild MCI, and cognitively normal groups respectively (De Meyer et al., 2010). The cognitively normal group with an AD signature were enriched in apolipoprotein E4 alleles. Validation of these findings in two further data sets showed that 64/68 (94% sensitivity) of autopsy-confirmed AD patients were classified with an AD signature while 57 MCI patients followed for 5 years had a sensitivity of 100% in pro‐ gressing to AD based on their biomarker signature. The presence of a CSF AD signature in cognitively normal subjects was interpreted by the authors as an indication of AD pathology being present and detectable far earlier than previously envisioned in disease progression.

Neurofilaments (NFs) are neuron-specific intermediate filaments and serve as a major cytos‐ keletal component in neurons. In a mature mammalian neuron, NFs are co-assembled from three subunits, termed NF-H (high), NF-M (medium) and NF-L (low). As NFs are confined to the nervous system, they might be one of the best markers reflecting neuronal pathogenic changes seen in some neurological disorders, such as AD. In AD brain, the levels of phos‐ phorylated NF-H/M (pNF-H/M) have been found to be markedly increased (Wang et al., 2001). Hu et al., (2002) found that, the levels of phosphorylated NF-H/M (pNF-H/M), nonphosphorylated NF-H/M (npNF-H/M) and NF-L were significantly higher (pNF-H/M,,12–24 fold; npNF-H/M,,3–4-fold) in neurologically healthy aged people than young individuals. In AD, the levels of npNF-H/M, and NF-L were similar to vascular dementia (VaD), and higher than in age-matched controls and the levels of pNF-H/M were significantly higher AD and ALS than in aged controls and VaD. Based on these findings, it is suggested that the increased level of total NF, p-NF proteins in CSF could be used as a marker for brain aging and neuro‐ degenerative disorders in general, and the levels of pNF-H/M as a marker to discriminate AD from normal brain aging and as well as neurological conditions including VaD (Hu et al 2002).

Specific antibodies derived from aberrantly and hyperphosphorylated neuronal intermediate

filament peptides from AD brain as bio markers for early AD detection

between CSF levels and duration or severity of AD (Andreasen et al., 1999b).

**2.4. Combination of CSF amyloid and tau phosphorylation**

**2.5. NF proteins**

200 Understanding Alzheimer's Disease


**Table 5.** Phosphopeptides and phosphorylation sites identified in NF-M and NF-H


proteins, neurofilaments, MAP2 and Vimentin, should provide better understanding the biology and progression of the disease as well as provide additional biomarker at the early

Candidate Bio-Markers of Alzheimer's Disease

http://dx.doi.org/10.5772/55069

203

As the AD signature approach based on the amyloid and tau causality hypothesis of AD continues to evolve, other CSF biomarkers are also being assessed. These include CSF cytokines (Swardfager et al.,2010; Olson etal.,2010 )– specifically TGFβ increases in AD CSF (Swardfager et al.,2010)– CSF proteomic profiles (Papassotiropoulos et al.,2006), clusterin (Thambisetty et al.,2010)and IgG antibodies from the adaptive immune system (Reddy et al.,2011) The latter is a field of intense research, despite the challenges in analyzing proteome profiles, and involves the study of differences in the CSF proteome in AD, MCI and control subject groups (Papassotiropoulos et al.,2006;Zhang et al.,2005; Castano et al 2006; Finehout et al.,2007; Marouf et al.,2009; Choi et al.,2010). One study (Maarouf et al.,2009) reported changes in a variety of CSF proteins including a-2-macroglobulin, α1-antichymotrypsin,a1-antitrypsin, complement and heat shock proteins, cathepsinD, enolase and creatine. The ADNI is also generating CSF proteomic profiles as part of its ''Use of Targeted Multiplex Proteomic Strategies to Identify Plasma-Based Biomarkers in Alzheimer's Disease'' (Miller et al., 2009).

**3. Oxidized proteins: Potential candidate biomarkers in AD**

Although the pathogenesis of AD is not yet fully known, it is clear that the disease is caused by a combination of risk factors. Among several hypotheses, oxidative stress is considered to play a significant role (Butterfield, 2007). Although CSF represents the most suitable biological fluid to study neurodegenerative diseases since it can reflect the biochemical changes occurring in brain, its analysis is not always easily feasible for a large scale screening, because the costs involved are enormous and procedures are invasive, uncomfortable and not without risk. For a full screening and early diagnosis, biomarkers easily detectable in biological samples, such as plasma, are needed. Up to now, the search for reliable biomarkers for AD in peripheral blood is very challenging because of difficulties with the standardization of the methods of analysis and the low reproducibility of the results. Although a set of plasma markers that differentiated AD from controls have been shown to be useful in predicting conversion from MCI to AD (Song., 2009), the study has not been yet verified by other researchers and the application of these candidate biomarkers have yet to achieve the diagnostic power, sensitivity, and reproducibility necessary for widespread use in a clinical setting. Oxidized proteins may

represent potential candidate biomarkers for "oxidative stress diseases", such as AD.

The first report on protein oxidation in CSF samples was from Tohgi et al. (1999) who dem‐ onstrated that 3-nitrotyrosine moderately but significantly increased with advancing age, and showed a remarkable increase in patients with AD. As the free tyrosine concentration did not decrease, the increase in 3-nitrotyrosine with age or associated with AD did not appear to be directly related to an increase in free-nitrated tyrosines. Rather, the increased 3-nitrotyrosine

stage of the disease.

**2.7. Other CSF biomarkers for AD**

**Table 6.** Phosphopeptides and phosphorylation sites identified in MAP1 and MAP2

#### **2.6. Microtubule-associated proteins and vimentin**

Microtubules are polymers of α- and β-tubulin dimers that mediate many functions in neurons, including organelle transport and cell shape establishment and maintenance as well as axonal elongation and growthcone steering in neurons. The polymerization, stabilization, and dynamic properties of microtubules are influenced by interactions with microtubule-associ‐ ated proteins (MAPs). Members of this protein family are classified by size: high molecular mass proteins (MAP1A, MAP1B, MAP2a, and MAP2b) and intermediate molecular mass MAPs (MAP2c, MAP2d, and tau) (Gonzalez-Billault,C et al.,2004).

Increasing evidence highlights the critical outcome of MAP modification in cytoskeletal disorganization associated with the early stages of AD development. A decreased content of MAP1B and tau associated with cytoskeletal breakdown was found in the brains of AD patients compared with those of control individuals, suggesting a decreased capacity of microtubule assembly and stability (Nieto,A et.al 1989). These results are consistent with those of Iqbal et al. (1986) describing a decreased capacity in the in vitro microtubule assembly from brain extracts of AD patients. One study has shown an early decrease in MAP2 labeling within dendrites from AD brain (Adlard, P. A., and Vickers, J. C. 2002). Other studies have demon‐ strated that MAP1B and MAP2 co-localize with NFTs (Kosik et al., 1984; Takahashi, et al., 1991). Alonso et al. (1997) studied the associations of the Alzheimer-hyperphosphorylated tau (AD P-tau) with the high molecular weight MAPs (HMW-MAPs) MAP1 and MAP2. The author found that AD P aggregate with MAP1 and MAP2. The association of AD P-tau to the MAPs resulted in inhibition of MAP-promoted microtubule assembly. These studies suggest‐ ed that the abnormally phosphorylated tau can sequester both normal tau and HMW-MAPs and disassemble microtubules.

Vimentin is a 57-kDa intermediate filament (IF) protein commonly found in mesodermally derived cells. In the healthy adult brain, vimentin is lacking in neurons and generally restricted to vascular endothelial cells and certain subpopulations of glial cells at specific brain locations. Eli et al (2009) found that Vimentin was localized to neuronal perikarya and dendrites in AD brain, with vimentin-immunopositive neurons prevalent in regions exhibiting intra- and extracellular beta-amyloid1-42 (Aβ42) deposition. Neuronal colocalization of vimentin and Aβ42 was common in the cerebral cortex, cerebellum and hippocampus (Eli et al., 2009). Our lab recently discovered that the protein tangles which are a hallmark of the disease involve at least three different proteins rather than just one (table 4-6). The discovery of these additional proteins, neurofilaments, MAP2 and Vimentin, should provide better understanding the biology and progression of the disease as well as provide additional biomarker at the early stage of the disease.

#### **2.7. Other CSF biomarkers for AD**

**MAP Sequence Phosphorylation site**

MAP2 KIDLS\*HVTS\*KCGS\*LK Ser1702, Ser1706

Microtubules are polymers of α- and β-tubulin dimers that mediate many functions in neurons, including organelle transport and cell shape establishment and maintenance as well as axonal elongation and growthcone steering in neurons. The polymerization, stabilization, and dynamic properties of microtubules are influenced by interactions with microtubule-associ‐ ated proteins (MAPs). Members of this protein family are classified by size: high molecular mass proteins (MAP1A, MAP1B, MAP2a, and MAP2b) and intermediate molecular mass

Increasing evidence highlights the critical outcome of MAP modification in cytoskeletal disorganization associated with the early stages of AD development. A decreased content of MAP1B and tau associated with cytoskeletal breakdown was found in the brains of AD patients compared with those of control individuals, suggesting a decreased capacity of microtubule assembly and stability (Nieto,A et.al 1989). These results are consistent with those of Iqbal et al. (1986) describing a decreased capacity in the in vitro microtubule assembly from brain extracts of AD patients. One study has shown an early decrease in MAP2 labeling within dendrites from AD brain (Adlard, P. A., and Vickers, J. C. 2002). Other studies have demon‐ strated that MAP1B and MAP2 co-localize with NFTs (Kosik et al., 1984; Takahashi, et al., 1991). Alonso et al. (1997) studied the associations of the Alzheimer-hyperphosphorylated tau (AD P-tau) with the high molecular weight MAPs (HMW-MAPs) MAP1 and MAP2. The author found that AD P aggregate with MAP1 and MAP2. The association of AD P-tau to the MAPs resulted in inhibition of MAP-promoted microtubule assembly. These studies suggest‐ ed that the abnormally phosphorylated tau can sequester both normal tau and HMW-MAPs

Vimentin is a 57-kDa intermediate filament (IF) protein commonly found in mesodermally derived cells. In the healthy adult brain, vimentin is lacking in neurons and generally restricted to vascular endothelial cells and certain subpopulations of glial cells at specific brain locations. Eli et al (2009) found that Vimentin was localized to neuronal perikarya and dendrites in AD brain, with vimentin-immunopositive neurons prevalent in regions exhibiting intra- and extracellular beta-amyloid1-42 (Aβ42) deposition. Neuronal colocalization of vimentin and Aβ42 was common in the cerebral cortex, cerebellum and hippocampus (Eli et al., 2009). Our lab recently discovered that the protein tangles which are a hallmark of the disease involve at least three different proteins rather than just one (table 4-6). The discovery of these additional

MAP1B VLSPLRS\*PPLIGSESAYESFLSADDK Ser1274 MAP1B VLSPLRS\*PPLIGSESAYESFLSADDK Ser1270 MAP1B VLS\*PLRSPPLIGSESAYESFLSADDK Ser1270

MAP2 VAIIRT\*PPKSPATPK Thr350

**Table 6.** Phosphopeptides and phosphorylation sites identified in MAP1 and MAP2

MAPs (MAP2c, MAP2d, and tau) (Gonzalez-Billault,C et al.,2004).

**2.6. Microtubule-associated proteins and vimentin**

202 Understanding Alzheimer's Disease

and disassemble microtubules.

As the AD signature approach based on the amyloid and tau causality hypothesis of AD continues to evolve, other CSF biomarkers are also being assessed. These include CSF cytokines (Swardfager et al.,2010; Olson etal.,2010 )– specifically TGFβ increases in AD CSF (Swardfager et al.,2010)– CSF proteomic profiles (Papassotiropoulos et al.,2006), clusterin (Thambisetty et al.,2010)and IgG antibodies from the adaptive immune system (Reddy et al.,2011) The latter is a field of intense research, despite the challenges in analyzing proteome profiles, and involves the study of differences in the CSF proteome in AD, MCI and control subject groups (Papassotiropoulos et al.,2006;Zhang et al.,2005; Castano et al 2006; Finehout et al.,2007; Marouf et al.,2009; Choi et al.,2010). One study (Maarouf et al.,2009) reported changes in a variety of CSF proteins including a-2-macroglobulin, α1-antichymotrypsin,a1-antitrypsin, complement and heat shock proteins, cathepsinD, enolase and creatine. The ADNI is also generating CSF proteomic profiles as part of its ''Use of Targeted Multiplex Proteomic Strategies to Identify Plasma-Based Biomarkers in Alzheimer's Disease'' (Miller et al., 2009).
