Determination of CSF Biomarkers in Alzheimer Disease

By Eurofins Medinet

Alzheimer’s disease (AD) is the leading cause of dementia among people aged 65 and older. The disease is characterized by a slow progressive loss of cognition, behavioral changes and loss of independence that ends in the complete destruction of the personality (Arnold et al, 1991). According to the latest estimates, the World global prevalence of AD is predicted to quadruple to 106 million by 2050. The Alzheimer’s Association today reports that in 2007 there are now more than 5 million people in the United States living with Alzheimer’s disease. This number includes 4.9 million people over the age of 65 and between 200,000 and 500,000 people under age 65 with early onset Alzheimer’s disease and other dementias. This is a 10% increase from the previous prevalence nationwide estimate of 4.5 million. By mid-century, the number of people with Alzheimer’s is expected to grow to as many as 16 million. The greatest risk factor for Alzheimer’s is increasing age. The direct and indirect costs of Alzheimer’s and other dementias amount to more than $148 billion annually.

The National Institute of Aging has initiated the Alzheimer's Disease Neuroimaging Initiative (ADNI), a large observational study of patients with AD, patients with mild cognitive impairment (MCI) and cognitively normal volunteers to assess longitudinal changes in AD. Because cognitive measures do not easily correlate disease-modifying effects of treatment, current trials of investigational compounds require large sample sizes and long treatment duration. Therefore the use of biomarkers in such trials can help in the understanding of disease progression and drug effect.

On a biological point of view, AD is characterized by the accumulation of extracellular senile plaques (mainly constituted of b-amyloid peptide [Ab] and Ab associated proteins), and intracellular neurofibrillary tangles [NFTs] (rich in abnormally phosphorylated tau [t] and ubiquitin |ub]) in cortical and limbic brain regions. The Ab in plaques arises from specific processing of amyloid precursor protein that is positively regulated by the presenilins PS1 and PS2 (De Strooper et al, 1998; Wolfe et al, 1999). PS1 and PS2 have been shown to be substrates of the Ub-proteasome system (Kim et al, 1997; Steiner et al, 1998; Marambaud et al, 1998). Although certain mutations in the genes encoding amyloid precursor protein and PS1/PS2 are known to confer a genetic predisposition to AD, such mutations are absent in the majority of patients with AD. The Ub-proteasome system was first implicated in AD through the immunohistochemical observation of Ub-conjugated t protein in neurofibrillary tangles (Morishima-Kawashima et al, 1993; Lowe and Mayer, 1993). Van Leeuwen et al (1998) discovered that a mutant form of Ub, termed Ub⁺¹, is selectively expressed in the brains of AD patients. Lam et al (2000) demonstrated a mechanism by which Ub⁺¹ expression could lead to dominant inhibition of the Ub proteasome system.

Biomarkers allow new therapies to be developed more quickly and can be directly associated with a drug treatment as companion diagnostic. Since a clinical diagnosis of Alzheimer disease is inaccurate even among experienced investigators in about 10% to 15% of cases, biomarkers might improve the accuracy of diagnosis. Indeed it is mandatory in a diagnosis to exclude other forms of dementia as fronto-temporal lobe degeneration (FTLD), dementia with Lewy bodies (DLB) and vascular dementia (VaD). In addition biomarkers might also serve as indirect measures of AD severity and could help to follow the evolution of the disease during a drug treatment. There are two directions in developing biomarkers of AD: (i) neuroimaging techniques which provide both structural and metabolic information about the brain, (ii) and CSF biomarkers which correlate the intensity of the disease. 

In this editorial, we summarize the research work reporting on CSF biomarkers in AD which are used in clinical trials. Lumbar punctures are used on a routine basis for decades.  Two large studies of lumbar punctures performed as part of an evaluation of possible AD biomarkers have shown that the procedure can be applied broadly and is well tolerated with low incidence of complication such as headache (Blennow et al, 1993; Andreasen et al, 2001). With the growing number of CSF biomarkers available and since the volume of CSF sample is limited, simultaneous analyses of parameters must be done: the use of bead microarrays (multiplexing) using the flow cytometric-based Luminex xMAP® technology (Luminex Corp., Austin, TX) allows such approach. 

Main CSF Biomarkers in AD

1-Aβ1-42
Aβ, and in particular Aβ1-42, has been studied frequently as a biomarker for AD. Significant decreased concentrations of CSF Aβ1-42 were found in AD patients compared to controls (Tamaoka et al, 1997 ; Shoji  et al, 1998 ; Andreasen et al, 1999 ), patients with VaD (Andreasen et al, 2001), and patients with FTLD (Riemenschneider et al, 2002). CSF concentrations in AD of Aβ1-42 were reduced by 40% to 50%, whereas concentrations of Aβ1-40 are similar to those of age-matched controls. CSFAβ1-42 did correlate to an extent with dementia severity; however, in most studies concentrations did not vary over intervals as long as 12 months. Mean CSF Ab42 levels were decreased in patients with DLB compared to normal subjects, but no differences were found between the AD and the DLB group (Kanemaru et al, 2000).

Plasma concentrations of Aβ1-42 cannot be used as a biomarker of AD; indeed plasma Aβ1-42 levels did not correlate with those in CSF (Mehta et al, 2001). Longitudinal studies have not shown a consistent change in plasma Aβ over time in AD patients (Mayeux et al, 2003) and cross sectional differences between AD patients and controls that would allow plasma Aβ concentrations to be used as a diagnostic measure have not been identified.

2-Total t protein (t-t)
CSF t has also been studied as a potential biomarker in AD. The level of CSF total t protein (t-t), which includes both normal and hyperphosphorylated t (P-t), seems to correlate with the number of NFTs in AD postmortem brains (Tapiola et al, 1997). Mean CSF t-t levels were found to be significantly elevated in AD patients as compared to control subjects (Kanemaru et al, 2000: Shoji  et al, 1998; Andreasen et al, 2001), FTLD patients (Mecocci et al, 1998; Riemenschneider et al, 2002), VaD patients (Mecocci et al, 1998; Andreasen et al, 2001), and DLB patients (Kanemaru et al, 2000; Andreasen et al, 2001).  In AD patients, increases of 2- to 3-fold of CSF t-t levels are classically observed. Weak correlations were present with changes in cognitive scores, and CSF t-t levels remain stably elevated in AD over time intervals of 12 months or longer. 

3-Hyperphosphorylated t (P-t)
P-t appears as another promising biomarker in view of the AD diagnostic.  Protein t may be phosphorylated at various sites of phosphorylation (P-t ₁₈₁, ₁₉₉, ₂₃₁, ₂₃₅, ₃₉₆, and ₄₀₄). Among these P-t, three can be correlated with the type of NFT involved : (i) pre-neurofibrillary tangles (P Thr₂₃₁-t); (ii) intra-neuronal neurofibrillary tangles (P Thr₃₈₁-t);  and (iii) extra-neuronal neurofibrillary tangles (P Ser₁₉₉-t) (Augustinack et al, 2002). All three P-t were elevated in the CSF of AD patients. These three measures have similar sensitivity, although P Thr₂₃₁-t may have somewhat greater specificity for AD versus other forms of dementia. Measurement of P Thr₂₃₁-t in CSF improved discrimination of AD from FTLD (Buerger et al, 2002) and measurement of PThr₁₈₁-t levels enhanced the discrimination of AD from DLB (Parnetti et al, 2001; Hampel et al, 2004; Vanderstichele et al, 2006).

Multiplex assay of Aβ1-42, t-t and P-t

In a recent work, Lewczuk et al (2008) evaluated the clinical performance of APOE genotyping and Aβ1-42, t-t and  PThr₁₈₁-t in a prospective multicenter study using the INNO-BIA AlzBio3 assay applied on Luminex platform. This work extends the data obtained by Olsson et al. (2005) and Vanderstichele et al. (2005) who first used multiplexing technology for measuring CSF biomarkers in dementia.

The Luminex xMAP technology (Luminex Corporation, Austin, TX, USA) is based on the coupling of capture antibodies to microspheres (with a unique color code based on a gradient of two dyes) and fluorescent dye-labeled detecting antibodies. By using a flow cytometry-based detector, multiple analytes can be measured simultaneously. Compared with ELISA, bead microarray technology requires less total assay time, fewer procedural steps and a smaller sample volume.

4-Isoprostanes
Isoprostanes are biomarkers of oxidative stress and can be measured with high-throughput, semi-automated LC-MS/MS assays (Haschke et al., 2007). Isoprostane levels were found to be markedly elevated in both frontal and temporal cortex of AD brains, but not in the corresponding areas of FTLD brains and controls (Practico et al, 1998; Yao et al, 2003). CSF F-2 isoprostane levels were found to be significantly increased in AD in comparison with FTLD patients (Grossman et al, 2005). In addition it appeared that the levels were highly correlated with the severity of the disease (Montine et al, 2002). Interestingly, Montine et al (2001) found that the levels of CSF F2-isoprostane associated with Aβ1-42 and t-t levels resulted in a sensitivity of 84% and a specificity of 89%.

5-Cytokines
AD senile plaques are associated with local inflammatory process involving microglia and astrocytes. Among the inflammatory mediators, several CSF cytokines such as interleukins (IL): IL-1b, IL-6, IL-12, IL-15 and Tumor Necrosis Factor TNFa have been determined. The results are giving conflicting data e.g. IL-6 was found to be decreased, increased or unchanged in comparison with control (Franck et al, 2003). Similar conclusion can be drawn for other cytokines (rev. in Teunissen et al, 2002).

Therefore although CSF levels of cytokines in AD and other dementias appear different from controls, their measurements cannot be used as biomarkers for differentiating between various types of dementias.

6-Other Biomarkers
None of other potential CSF biomarkers described in AD (such as apolipoprotein E, amyloid precursor protein (APP), glial fibrillary acidic protein, S-100B protein, alpha-1-antichymotrypsin, C-reactive protein, complement C1q, homocysteine, 3-nitrotyrosine, neuronal thread protein, NSE, ubiquitin and growth-associated protein-43) demonstrated to be useful in clinical practice. They are either yielding conflicting data between different studies or giving a lower capacity to discriminate ad from other dementias.

The use of proteomic techniques [such as two-dimensional gel electrophoresis (2D-GE) combined with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), mass spectrometry (MS) or surface-enhanced laser desorption/ionization (SELDI-)TOF MS] will certainly allow the identification of new AD potential biomarkers.

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