Amyloid-beta and tau proteins as biochemical markers of Alzheimer's disease

With the development of new therapeutic strategies, and the concept of mild cognitive impairment (MCI) as an early stage of Alzheimer's disease (AD), there is an increasing need for an early and accurate diagnosis of sporadic AD. Therefore, biological markers allowing a positive diagnosis early in the course of the disease are highly desirable. The most extensively evaluated markers of sporadic AD are amyloid-beta proteins and levels of both total and phosphorylated microtubule-associated protein tau. In this study, we review the currently available data on the aforementioned markers assessed in the cerebrospinal fluid or plasma, alone and in combinations, focusing on their clinical applicability including sensitivity in the diagnosis of AD and mild cognitive impairment, specificity in discriminating AD from other dementias and correlations with the disease progression and apolipoprotein E genotype. We also analyze advantages and potential drawbacks of using biomarkers in the laboratory diagnosis of AD.     

Monoamine oxidase B in brains from patients with Alzheimer's disease: a biochemical and autoradiographical study.

In vitro quantitative autoradiography using [3H]L-deprenyl, an irreversible and preferential inhibitor of monoamine oxidase B, was performed to investigate the localization of the enzyme in brains from senile dementia of Alzheimer type and control cases. Brains from three male patients with the clinical diagnosis of senile dementia of Alzheimer type and from three male control patients, without any known clinical history of neurological disorder, were obtained at autopsy. Cryosections of 100 microns thickness were mounted on gelatinized glass plates and dried over desiccant for one week at -20 degrees C. The sections were incubated with 10 nM [3H]L-deprenyl for 1 h and then exposed to film for four weeks. The autoradiographs were analysed by computer-assisted densitometry. Monoamine oxidase-B activities were also estimated in 1% homogenates from 10 different regions, using 10 microM beta-[ethyl-14C]phenylethylamine, in order to study the consonance between the autoradiographical and biochemical techniques. Both [3H]L-deprenyl binding and monoamine oxidase-B activities in senile dementia of Alzheimer type were higher than in the controls in all brain regions studied. The increase was highest in the white matter (about 70%) and in the order of 20-50% in the various gray matter regions. A high correlation coefficient (r approximately 0.9) was obtained between [3H]L-deprenyl binding and monoamine oxidase-B activity, both in the senile dementia of Alzheimer type and in the control brains.     

Tissue transglutaminase is not a biochemical marker for Alzheimer's disease

Typical hallmarks of Alzheimer's disease (AD) are pathologic deposits in cortical and subcortical regions consisting of self-aggregated proteins such as amyloid-beta (Aβ) or tau. Tissue transglutaminase (tTG) catalyses calcium-dependent cross-linking between proteins (transamidation) resulting in protease-resistant isopeptide bonds. Because of this ability, tTG was suspected to participate in AD pathogenesis. Aβ and tau can be cross-linked by tTG in vitro. In AD neocortex, messenger RNA expression of tTG is increased. However, data on transamidation in AD specimens—activity of not only tTG but also other transglutaminases—are contradictory. The aim of our study was to investigate if tTG is involved in AD development and may be useful as biomarker for AD. We studied human brain samples for tTG concentration, tTG localization, and transamidation activity and cerebrospinal fluid (CSF) for tTG content by novel sensitive and highly specific methods. Neither tTG concentration nor transamidation was increased in AD brain homogenates. Immunohistologically, we found no colocalization of tTG in neocortex sections with tau or Aβ deposits but with blood vessels. Only in rare cases, tTG was detectable in CSF samples. This could be attributed to liberation from erythrocytes. Our data contradict the view that tTG is a potential biochemical marker for AD.     

Diagnosing prodromal Alzheimer's disease: role of CSF biochemical markers

Mild cognitive impairment (MCI) is an aetiologically heterogeneous syndrome. A correct prediction of MCI conversion to Alzheimer's disease (AD) represents a primary goal in routine clinical practice. Since the presence of pathological levels in >or=2 cerebrospinal fluid (CSF) biomarkers; amyloid protein (Abeta42), total tau (h-tau) and phospho-tau (p-tau) seems to reliably identifying MCI subjects converting to AD, we report our experience in a routine clinical setting. In the period from January 2001 to June 2003, 273 consecutive patients referred to our Memory Clinic for diagnostic assessment of cognitive impairment. Of them, 180 underwent a complete diagnostic evaluation including CSF dosage of fragment 1-42 of amyloid protein, total tau and phospho-tau (ELISA Method, Innogenetics, Gent, Belgium), after vascular or other secondary causes of dementia could be excluded. At baseline, 38% of the MCI subjects (20/55) showed pathological levels in >or=2 CSF biomarkers. After 1 year, 11 MCI patients converted to dementia, 33 remained stable, 11 showed a further progression of cognitive impairment still not fulfilling the diagnostic criteria for dementia. Of the 11 converters, 10 showed >or=2 pathological values CSF biomarkers and in all of them, p-tau was high. On the contrary, 29 out of 33 stable MCI (88%) showed no or one pathological CSF value. We confirm that pathological levels in >or=2 CSF biomarkers reliably predict MCI conversion to AD and correctly identify the stable form of MCI.     

Plasma osteoprotegerin as a biochemical marker for vascular dementia and Alzheimer's disease

Elevated level of osteoprotegerin (OPG), a pleiotropic cytokine involved in bone metabolism, has been associated with coronary heart disease and higher cardiovascular mortality. Because cardiovascular disorders are recognized risk factors for dementia, the study of OPG as a disease marker in vascular dementia (VaD) and Alzheimer's disease (AD) seemed worthy of investigation. OPG concentration was determined by ELISA in an Italian cohort consisting of 39 VaD patients, 36 AD patients, and 39 non-demented controls strictly matched for age and gender. Plasma OPG levels were positively related to age in both demented and non-demented persons. OPG concentrations were significantly higher in both VaD (median: 4.75 pmol/l; interquartile range: 3.42-6.85 pmol/l; P<0.0001) and AD (median: 4.02 pmol/l; interquartile range: 3.07-4.77 pmol/l; P=0.0278) compared to non-demented controls (median: 3.24 pmol/l, interquartile range: 2.70-3.98 pmol/l). After allowance for confounding factors (age, gender and APOE epsilon4 allele), plasma OPG levels remained independently associated with the presence of VaD (OR = 2.51; 95% CI 1.46-4.32; P=0.0009) and AD (OR = 2.17; 95% CI 1.18-3.99; P=0.0126). Our study demonstrates that OPG may be regarded as a novel biomarker of dementia in the Italian population. These results further support the hypothesis that vascular factors may not only play a role in the pathogenesis of VaD but also in the pathogenesis of AD.     

Complexity analysis of the magnetoencephalogram background activity in Alzheimer's disease patients

The aim of the present study was to analyse the magnetoencephalogram (MEG) background activity in patients with Alzheimer's disease (AD) using the Lempel-Ziv (LZ) complexity. This non-linear method measures the complexity of finite sequences and is related to the number of distinct substrings and the rate of their occurrence along the sequence. The MEGs were recorded with a 148-channel whole-head magnetometer (MAGNES 2500 WH, 4D Neuroimaging) in 21 patients with AD and in 21 age-matched control subjects. Artefact-free epochs were selected for complexity analysis. Results showed that MEG signals from AD patients had lower complexity than control subjects' MEGs and the differences were statistically significant (p<0.01). In order to reduce the dimension of the LZ complexity results, a principal components analysis (PCA) was applied, and only the first principal component was retained. The first component score from PCA was graphically analysed using a box plot and a receiver-operating characteristic (ROC) curve. A specificity of 85.71%, a sensitivity of 80.95% and an area under the ROC curve of 0.9002 were obtained. These preliminary results suggest that cognitive dysfunction in AD is associated with a decreased LZ complexity in the MEG signals.     

Chemotactic-like receptors and Abeta peptide induced responses in Alzheimer's disease

Evidence suggests that beta-amyloid (Abeta) has chemokine-like properties and may act through formyl chemotactic receptors (FPR) to induce pathophysiologically important functional changes in Alzheimer's disease (AD) microglia. We have shown that Abeta 1-42, fibrillar Abeta 1-40, and Abeta 25-35 potentiate the release of interleukin-1beta (IL-1beta) from LPS activated human THP-1 monocytes [26] and LPS primed rat microglia. Moreover, Abeta-stimulated IL-1beta secretion seems to be receptor mediated because it is calcium dependent and requires activation of specific G-proteins [27]. Thus, we have evaluated the ability of Abeta 1-42 to mimic formyl chemotactic peptides in stimulating IL-1beta release from THP-1 monocytes. Several of the formyl chemotactic peptides and Abeta 1-42 significantly enhanced IL-1beta production in THP-1 monocytes. In contrast, a formyl chemotactic receptor antagonist inhibited Abeta 1-42-induced IL-1beta release from both human THP-1 monocytes and primary rat microglia. Further, primary rat microglia grown in culture expressed FPR as demonstrated by immunocytochemistry. Given the multiple pathophysiologic roles IL-1beta may play in AD, agents that block Abeta interactions with formyl chemotactic receptors on microglia might be important antiinflammatory therapeutic targets.     

Fractal analysis of amyloid plaques in Alzheimer's disease patients and mouse models

The varied morphological and biochemical forms in which amyloid deposits in brain of Alzheimer's disease (AD) patients are complex and their mechanisms of formation are not completely understood. Here we investigated the ability of fractal dimension (FD) to differentiate between the textures of commonly observed amyloid plaques in sporadic and familial AD patients and aged-control individuals as well as in transgenic mouse models of amyloidosis. Studying more than 6000 amyloid plaques immunostained for total Aβ (Aβt), Aβ40 or Aβ42, we show here that Aβ40 FD could efficiently differentiate between (i) AD patients and aged-control individuals (P < 0.001); (ii) sporadic and familial AD due to presenilin-1 or APP (A692G) mutations (P < 0.001); and (iii) three transgenic mouse models of different genotypes (P < 0.001). Furthermore, while diffuse and dense-core plaques present in humans and transgenic mice had comparable FDs, both Aβt and Aβ42 FD could also differentiate diffuse plaques from other plaque types in both species (P < 0.001). Our data suggest that plaque FD could be a valuable tool for objective, computer-oriented AD diagnosis as well as for genotype-phenotype correlations of AD.     

Candidate gene analysis of IP-10 gene in patients with Alzheimer's disease

Interferon-gamma-inducible Protein-10 (IP-10) is supposed to play a role in Alzheimer's disease (AD) development, as demonstrated by increased levels in cerebrospinal fluid from patients with AD. A mutation scanning of IP-10 exonic region was carried out in 10 patients with AD and 10 age-matched controls, demonstrating the presence of two previously reported single nucleotide polymorphisms (SNPs) in exon 4 (G-->C and T-->C) as well as a novel SNP in exon 2 (C-->T). Exon 4 G-->C and T-->C allelic variants were next evaluated in a population of 279 AD patients and 251 controls, in order to determine whether their presence could influence the susceptibility towards the development of the disease. These two SNPs were in complete linkage disequilibrium. No differences in haplotype frequencies were found in AD patients as compared with controls, even stratifying according to the presence of Apolipoprotein E varepsilon4 allele, gender or age at onset. A new protocol was developed to easily determine the C-->T SNP in exon 2. A preliminary analysis revealed a very low frequency of this allelic variant (1%). Therefore, the complete association study was not carried out because the size of our population was not sufficient to draw reliable conclusions. According to these results, IP-10 does not seem to be a risk factor for AD. However, a novel rare polymorphism has been identified, which could exert a role in AD susceptibility. Thus, further studies on larger populations are needed before confidently excluding IP-10 as a susceptibility gene for AD.     

Mitochondrial DNA deletions cause the biochemical defect observed in Alzheimer's disease

Alzheimer's disease (AD) is the most common form of dementia, increasing in prevalence with age. Most patients who develop AD have an unknown cause, but characteristic neuropathological features include the deposition of extracellular amyloid beta and of intraneuronal hyperphosphorylated tau protein. Researchers have previously implicated mitochondrial dysfunction in AD. We previously showed an increase in neurons displaying a mitochondrial biochemical defect-cytochrome-c oxidase (COX) deficiency-in the hippocampus in patients with sporadic AD compared with age-matched controls. COX deficiency is well described as a marker of mitochondrial (mt) DNA dysfunction. This present study analyzed the mtDNA in single neurons from both COX normal and COX-deficient cells. Analysis of the mtDNA revealed that COX deficiency is caused by high levels of mtDNA deletions which accumulate with age. Future research is needed to clarify the role mtDNA deletions have in normal aging and investigate the relationship between mtDNA deletions and the pathogenesis of sporadic AD.     

Exploratory analysis of seven Alzheimer's disease genes: disease progression

The relationships between genome wide association study-identified and replicated genetic variants associated with Alzheimer's disease (AD) risk and disease progression or therapeutic responses in AD patients are almost unexplored. Seven hundred and one AD patients with at least 3 different cognitive evaluations and genotypic information for APOE and 6 genome wide association study-significant single-nucleotide polymorphisms were selected for this study. Mean differences in Global Deterioration Score and Mini Mental State Examination (MMSE) were evaluated using nonparametric tests, general linear model and mixed models for repeated measurements. Each chart was also reviewed for evidence of treatment with any cholinesterase inhibitor, memantine, or both. Relationships between therapeutic protocols, genetic markers, and progression were explored using stratified analysis looking for specific effects on progression in each therapeutic category separately. Neither calculation rendered a Bonferroni-corrected statistically significant difference in any genetic marker. Mixed model results suggested differences in the average point in MMSE test for patients carrying PICALM GA or AA genotype compared with GG carriers at the end of the follow-up (MMSE mean difference = −0.57; 95% confidence interval, −1.145 to 0.009; p = 0.047). This observation remained unaltered after covariate adjustments although it did not achieve predefined multiple testing significance threshold. The PICALM single-nucleotide polymorphism also displayed a significant effect protecting against rapid progression during pharmacogenetic assays although its observed effect displayed heterogeneity among AD therapeutic protocols (p = 0.039). None of the studied genetic markers were convincingly linked to AD progression or drug response. However, by using different statistical approaches, the PICALM rs3851179 marker displayed consistent but weak effects on disease progression phenotypes.     

Brain cholinesterases: I. The clinico-histopathological and biochemical basis of Alzheimer's disease

Substantial evidence is presented demonstrating that it is the cholinesterases (ChEs) that constitute the organizer, the connector and the safeguard for multiple neurochemical functions and mature anatomical architecture of the brain. In Alzheimer's disease (AD), the histopathological characteristics are initially and primarily associated with the degeneration of the acetylcholinesterase (AChE) system in various brain regions. Multiple classic and/or putative neurotransmitters and neuromodulators, virtually all the peptide hormones of the endocrine and neuroendocrine systems in the brain, their specific synthesizing and hydrolyzing marker enzymes and associated uptake processes (transporters), and receptors, do not actually participate in the formation of senile plaques and neurofibrillary tangles in the brains of patients suffering from AD. The massive perturbation in different neurochemicals seen in AD is essentially caused by the ChEs-associated pathology. The graded patterns of brain ChEs expression affect the preferential vulnerability and severity of the AD clinico-pathologic presentation. It seems that the common law in nature may also dominate the destiny of brain ChEs system, i.e., the weaker the cells express AChE, the more susceptible the cells are to AD degeneration, and vice versa.     

Hypothesis: Alzheimer's disease is a phylogenetic disease

It is hypothesized that Alzheimer's disease is a human phylogenetic disease which has a common multifactorial pathogenesis in sporadic and familial cases and in Down syndrome, related to a genomic character function G(x). Increments in G(x) accompanied the increased gene expression that sustained brain growth and differentiation during hominid evolution, particularly of the regions liable to Alzheimer pathology, and further occur in Down syndrome [suggesting that genes on chromosome 21 are included in G(x)]. If genes which promoted human brain evolution contribute to the value of G(x), a better understanding of the genomic events which promoted this evolution, using molecular biological techniques, should elucidate the genetic basis of Alzheimer's disease, and vice versa.     

Mitochondrial DNA sequence analysis of four Alzheimer's and Parkinson's disease patients

The mitochondrial DNA (mtDNA) sequence was determined on 3 patients with Alzheimer's disease (AD) exhibiting AD plus Parkinson's disease (PD) neuropathologic changes and one patient with PD. Patient mtDNA sequences were compared to the standard Cambridge sequence to identify base changes. In the first AD + PD patient, 2 of the 15 nucleotide substitutions may contribute to the neuropathology, a nucleotide pair (np) 4336 transition in the tRNA^Gln gene found 7.4 times more frequently in patients than in controls, and a unique np 721 transition in the 12S rRNA gene which was not found in 70 other patients or 905 controls. In the second AD + PD patient, 27 nucleotide substitutions were detected, including an np 3397 transition in the ND1 gene which converts a conserved methionine to a valine. In the third AD + PD patient, 2 polymorphic base substitutions frequently found at increased frequency in Leber's hereditary optic neuropathy patients were observed, an np 4216 transition in ND1 and an np 13708 transition in ND5 gene. For the PD patient, 2 novel variants were observed among 25 base substitutions, an np 1709 substitution in the 16S rRNA gene and an np 15851 missense mutation in the cytb gene. Further studies will be required to demonstrate a causal role for these base substitutions in neurodegenerative disease. © 1996 Wiley-Liss, Inc.     

Electroneuromyographic findings in patients with Alzheimer's disease

Electroneurophysiological abnormalities have been reported in some patients with dementia, but the data available is rather limited and controversial. We studied nerve conduction velocities and electromyography in 59 patients with moderate to severe involvement of Alzheimer's disease. Except some occasional mono- and polyneuropathies, the patients showed no systemic dysfunction of the peripheral nervous system.