Laboratory Diagnosis in Neurology, 1 Ed.

Markers of Dementia and Neuronal Destruction

M. Otto

Tau Protein

Physiology

Isoforms. Tau proteins are microtubule-associated proteins of low molecular weight, found primarily in the axons of CNS neurons. Human tau protein is coded by the tau gene located on chromosome 17. Alternative splicing of 11 out of 16 exons generates different tau isoforms. In the human brain, only six isoforms are found; they are between 352 and 441 amino acids long.

Function. The main function of tau is the stabilization of microtubules; it also promotes polymerization of microtubules. Tau binding to microtubules is influenced by phosphorylation, particularly near the microtubule-binding region of the isoforms. Up to 30 out of 79 possible phosphorylation sites of the amino acids serine and threonine have been described (Buee et al., 2000).

Pathophysiology

The idea of determining tau protein in the CSF was based on the fact that the intracellular neurofibrillary tangles found in the brains of patients with Alzheimer's disease consist predominantly of paired helical filaments composed of hyperphosphorylated tau isoforms. The degree and localization of phosphorylation seem to vary with different diseases (e. g., Alzheimer's disease and other tauopathies). Relatively little is known about the regulation of phosphorylation. There is debate as to whether 14–3-3 proteins play a role here as they were also found in neurofibrillary tangles (Layfield et al., 1996; Hashiguchi et al., 2000).

Interpretation

Alzheimer's disease. Elevated levels of total tau protein were first described in the CSF of patients with Alzheimer's disease in 1995, detected by ELISA (Jensen et al., 1995; Vigo Pelfrey et al., 1995). Initially, this elevation was thought to be specific to Alzheimer's disease. However, further studies showed elevated levels not only in these patients (Andreasen et al., 1999 b), but also in patients with other forms of dementia (see below). The predictive value of tau protein determination is therefore low.

Dementia vs. depression. Tau protein determination is appropriate for distinguishing between dementia and depression. The tau protein levels associated with different conditions are as follows:

• Healthy persons: below 450 pg/mL.

• Patients with Alzheimer's disease: between 450 pg/mL and 800 pg/mL.

• Patients with Creutzfeldt-Jakob disease (CJD): usually above 1300 pg/mL.

• Patients with variant Creutzfeldt-Jakob disease (vCJD): usually above 500 pg/mL, but below 1300 pg/mL.

Currently, several ELISA tests are available for determining specific isoforms of phospho-tau.

Differential diagnosis of dementia. The largest multicenter study carried out so far investigated whether neurochemical diagnosis of dementia can be improved by an ELISA test detecting the fraction of tau protein that is phosphorylated at serine position 199 (phospho-tau199) (Itoh et al., 2001). For this purpose, the CSF from 570 patients was examined for total tau as well as phospho-tau199. The patients were grouped as follows:

• Alzheimer's disease (n = 236).

• Frontotemporal dementia (n = 16).

• Corticobasal degeneration (n = 15).

• Progressive supranuclear paralysis (n = 15).

• Lewy body dementia (LBD) (n = 13).

• Vascular dementia (n = 23).

• CJD (n = 11).

The control group was composed of patients with various neurological diseases without dementia (n = 122) and patients without neurological or psychiatric disease (n = 95). Distinction between Alzheimer patients and those with other forms of dementia was successful with the total tau ELISA test; it had a sensitivity of 77% and a specificity of 78%. Patients with CJD showed the highest levels and were easy to distinguish from patients with other forms of dementia. Using phospho-tau199, Alzheimer's disease was distinguished from the other dementias with both a sensitivity and a specificity of 85%. Phospho-tau199 was not significantly elevated in patients with CJD. Studies employing other phospho-tau ELISA tests on equal-sized groups of Alzheimer patients and patients with Lewy body dementia were unable to identify clinically useful threshold values for the differential diagnosis (Mollenhauer et al., 2005). It remains to be seen whether phospho-tau determination will become established in the early or differential diagnosis of dementias.

Amyloid Beta (Aβ) Peptides

Physiology and Pathophysiology

Physiology. Amyloid beta (Aβ) peptides are generated through enzymatic cleavage of amyloid precursor protein (APP) by α-, β-, and γ-secretases and constitute the main ingredient of amyloid plaques in Alzheimer's disease (Glenner and Wong, 1984; Masters et al., 1985). These peptides form a heterogeneous group of 37–42 amino acids in length, with the Aβ peptide1–40 clearly dominating (Wiltfang et al., 2002). Aβ peptide1–42 differs from Aβ peptide1–40 by two additional neutral amino acids at the carboxyterminal end; its proportion under physiological conditions is only 10% of the total.

Pathophysiology. Although the primary sequences are almost identical, the longer Aβ peptide1–42 is less soluble than Aβ peptide1–40. It aggregates faster and forms neurotoxic β-amyloid depositions. These neuritic extracellular β-amyloid plaques consist predominantly of Aβ peptide1–42. Using ELISA, it is possible today to distinguish between Aβ peptide1–42 and Aβ peptide1–40 in the CSF.

Analysis

Procedure. Several ELISA tests are currently available for determining Aβ peptide1–42 and Aβ peptide1–40. Furthermore, some specialized laboratories perform a special Aβ immunoblot following urea gel electrophoresis which, in addition to Aβ peptide1–42 and Aβ peptide1–40, provides quantitative determination of other Aβ peptide fragments (Wiltfang et al., 2003).

Cut-Off Levels. The cut-off levels depend on the clinical condition in question and may vary among laboratories. Pathological levels of Aβ peptide1–42 are usually below 450 pg/mL.

Interpretation

Alzheimer's disease. Aβ peptide1–42 is significantly diminished in Alzheimer patients. In combination with the increase in tau protein, this finding is relatively characteristic of Alzheimer's disease (Andreasen et al., 1999 a; Hulstaert et al., 1999).

Studies with small case numbers have shown that Aβ peptide1–42 levels are already low in Alzheimer patients with “mild cognitive impairment.” A decrease in Aβ peptide1–42 may therefore be an early marker of Alzheimer's disease. Of pathophysiological interest, Aβ peptide1–42 may also be decreased in patients with CJD (Otto et al., 2000; Kapaki et al., 2001; Vanmechelen et al., 2001).

Differential diagnosis of dementia. A decrease in Aβ peptide1–42 was found not only in Alzheimer's disease, but also in other forms of dementia (e. g., Lewy body dementia, CJD). In the actual case, it may therefore be difficult to classify the patient.

Until now, the decrease in Aβ peptide1–42 in CSF was explained by its deposition in amyloid plaques, which thus lowered the CSF concentration. However, amyloid plaques were found in only some of the CJD patients examined. One suggestion is that, in amyloidosis (Alzheimer's disease and CJD), Aβ peptides bind with high affinity to specific chaperones and form high-molecular-weight complexes, which because of epitope masking can no longer be detected by the usual means (ELISA) (Wiltfang et al., 2003). For the differential diagnosis of CJD, this means that a drop in CSF Aβ peptide1–42 measured with ELISA cannot be used to exclude CJD.

Recent studies—again with relatively few cases—in which several Aβ peptide fragments were determined either with ELISA (Aβ peptide1–42/Aβ peptide1–40) or with the Wiltfang urea gel electrophoresis (Aβ peptides1–37/38/39/40/42) suggest that these determinations more successfully distinguish between the groups.

14–3-3 Proteins

Physiology

Isoforms.14–3-3 proteins are a family of regulatory proteins with a molecular weight of about 30 kDa. At least seven isoforms are known; they exist as dimers and have a highly conserved amino acid sequence in almost all eukaryotic species.

Function.14–3-3 proteins are thought to play a role in signal transduction, particularly in mediating the binding of kinases (Berg et al., 2003). 14–3-3 proteins are increasingly regarded as chaperones.

Analysis

Immunoblot. The antibody used for detection in immunoblots recognizes the N-terminal amino acid sequence common to all seven human isoforms (α to η). The monomers of physiologically occurring dimers are connected by means of this N-terminal, thus preventing recognition of the native 14–3-3 protein. This disadvantage is of minor importance for the detection in immunoblots because the dimeric 14–3-3 protein is first dissociated by sodium dodecyl sulfate (SDS), thus allowing detection of monomers.

ELISA. Two ELISA methods for 14–3-3 proteins have been presented, but found no general application.

Interpretation

The determination of 14–3-3 isoforms is of interest in the differential diagnosis of CJD. Two protein spots (P130 and P131) thought to be specific for CJD were detected by twodimensional polyacrylamide gel electrophoresis (2 D PAGE) in the CSF of CJD patients (Hsich et al., 1996). After partial sequencing of their amino acids, P130 was identified as member of the 14–3-3 protein family. It is not clear whether 14–3-3 proteins are pathologically important in CJD. The 14–3-3 isoform spectrum found in CSF is different from that in the brain. When 14–3-3-γ knockout mice were inoculated with pathological prion protein, their survival rate did not differ from that of control mice (Steinacker et al., 2005).

Several studies have confirmed the high diagnostic reliability of the SDS-PAGE immunoblot techniques currently used for 14–3-3 proteins in CSF (Zerr et al., 1998; Otto et al., 2002). Since then, patients who fulfill the clinical criteria of “possible” CJD and show positive CSF results are classified as “probable” CJD cases, irrespective of their EEG results. With vCJD, however, the SDS-PAGE immunoblot is not always positive.

Pitfalls in Determining 14–3-3 Proteins

False-positive results may occasionally occur in Alzheimer patients, in CSF altered by inflammation, after ischemic events, and in glioblastoma patients. It should be kept in mind, however, that these diseases can normally be differentiated on a clinical basis. Total tau protein determination provides a similarly clear distinction. The 14–3-3 immunoblot usually becomes positive with tau protein levels above 1100–1300 pg/mL.

S-100 Proteins in CSF

Physiology

Occurrence and isoforms. S-100 protein is an acidic calcium-binding protein with a molecular weight of 21 kDa occurring primarily in the nervous system of vertebrates (Schäfer and Heizmann, 1996). Native S-100 is found as homodimer or heterodimer with two isomeric subunits, α and β. According to the new nomenclature, the α-subunit is called S-100A, and the β-subunit is called S-100B (Schäfer et al., 1995). All three possible combinations occur. The isoforms have a molecular weight of 10.5 kDa each. S-100B (S-100 β/β) is found in high concentrations in glial cells, while S-100A (S-100 α/β) is found in glial cells excepting Schwann cells. S-100 occurs in much lower concentrations in peripheral tissue than in the central nervous system.

Function. Experiments suggest that S-100 functions as a neurotrophic factor.

Interpretation

Creutzfeldt-Jakob disease. Several studies have found elevated S-100 levels in CSF of patients with sporadic CJD and vCJD. The diagnostic sensitivity was 84% and the specificity 91%. When detection of S-100 in the serum was improved, or indeed made possible, by using a luminescence assay (Otto et al., 1998), it was shown that S-100B levels in serum were significantly higher in patients with CJD than in patients with other forms of dementia or in control persons without dementia. Using a cut-off point of 213 pg/mL, the diagnostic sensitivity was 78% and the specificity 81%. This sensitivity, however, is lower than that for 14–3-3 protein and tau protein in the CSF analysis. Nevertheless, it is evident that the S-100B levels in serum as well as in CSF increase during development of the disease, well before the complete clinical picture has developed. Since glial activation has also been described in Alzheimer's disease, it is currently under debate whether S-100B might be a suitable progression marker in the serum.

Other diseases. The diagnostic importance of S-100 protein is increasing not only as a tumor marker in malignant melanoma, but also as a prognostic indicator in ischemic cerebral infarcts (Missler et al., 1997; Herrmann et al., 2000), and in evaluating neuropsychological deficits after minimal craniocerebral trauma.

Neuron-Specific Enolase in CSF

Physiology. Neuron-specific enolase (NSE) is a 78-kDa glycolytic enzyme that is localized as a γ,γ-dimer in neurons and neuroendocrine cells. In the CSF, 98% of the protein stems from the brain (Jacobi and Reiber, 1986; this volume, Chap. 3, “Brain Proteins in the Blood,” and Table 3.4).

Interpretation. Pathological levels have so far been measured in serum and CSF of patients with hypoxemic brain damage, brain tumors, cerebral hemorrhage, and cerebral trauma (Schaarschmidt et al., 1994). In the differential diagnosis of dementia, NSE is gaining in importance as one of the first surrogate markers. The cut-off level for NSE in the CSF is 35 ng/mL; this permits diagnosis of patients as having CJD with a sensitivity of 78% and a specificity of 88%. With the assays used so far, no significantly different levels have been found in serum in CJD.

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