Juan Manuel Maler

Neurochemical diagnosis of Alzheimer’s dementia by CSF Aβ42, Aβ42/Aβ40 ratio and total tau

Cerebrospinal fluid (CSF) concentrations of amyloid β peptides ending at positions 42 and 40 (Aβ42 and Aβ40, respectively), and total tau (tTau) protein were measured by ELISA in order to compare their accuracy in discriminating patients with Alzheimer’s disease (AD, n=22), non-Alzheimer dementia (nAD, n=11) and control subjects (CON, n=35). As compared to the other groups, the concentrations of Aβ42 and tTau were decreased (P<0.001) and increased (P<0.001) in AD, respectively, while Aβ40 did not differ significantly among the groups. Receiver operating characteristic (ROC) analysis was performed to define cut-off values for maximized sensitivity and specificity. For all groups compared the Aβ peptide ratio 42/40 classified more patients correctly, as compared to the concentration of Aβ42 alone: AD versus controls, 94 and 86.7%; AD versus nAD, 90 and 85% and AD versus nAD plus controls, 90.8 and 87%, respectively. The percentage of correctly classified patients was further improved when the Aβ ratio was combined with the analysis of the tTau concentration. Presence of the apolipoprotein E e4 allele, age or degree of mental disability did not significantly influence the parameters studied.

Amyloid β peptide ratio 42/40 but not Aβ42 correlates with phospho-Tau in patients with low- and high-CSF Aβ40 load

Neurochemical dementia diagnostics (NDD) can significantly improve the clinically based categorization of patients with early dementia disorders, and the cerebrospinal fluid (CSF) concentrations of amyloid β peptides ending at the amino acid position of 42 (Aβx‐42 and Aβ1‐42) are widely accepted biomarkers of Alzheimer’s disease (AD). However, in subjects with constitutively high‐ or low‐CSF concentrations of total Aβ peptides (tAβ), the NDD interpretation might lead to erroneous conclusions as these biomarkers seem to correlate better with the total Aβ load than with the pathological status of a given patient in such cases. In this multicenter study, we found significantly increased CSF concentrations of phosphorylated Tau (pTau181) and total Tau in the group of subjects with high CSF Aβx‐40 concentrations and decreased Aβx‐42/x‐40 concentration ratio compared with the group of subjects with low CSF Aβx‐40 and normal Aβ ratio (p < 0.001 in both cases). Furthermore, we observed significantly decreased Aβ ratio (p < 0.01) in the group of subjects with APOE ε4 allele compared with the group of subjects without this allele. Surprisingly, patients with low‐Aβx‐40 and the decreased Aβ ratio characterized with decreased pTau181 (p < 0.05), and unaltered total Tau compared with the subjects with high Aβx‐40 and the Aβ ratio in the normal range. We conclude that the amyloid β concentration ratio should replace the ‘raw’ concentrations of corresponding Aβ peptides to improve reliability of the neurochemical dementia diagnosis.

International quality control survey of neurochemical dementia diagnostics.

UNLABELLED Currently, neurochemical dementia diagnostics (NDD) are increasingly entering routine clinical neurochemistry, offering improved early and differential diagnosis of dementias. However, there is an obvious lack of standardization in pre-analytical sample handling and systematic quality surveys. Therefore, in this study, 14 laboratories in Germany, Austria, and Switzerland were given aliquots of a human cerebrospinal fluid (CSF) sample, and were asked to measure Alzheimers disease (AD) biomarkers (amyloid beta (Abeta) peptides, total Tau protein, and phosphorylated Tau protein (P-tau(181P))) according to their routine protocols. RESULTS The inter-laboratory coefficients of variation of the results obtained by the laboratories participating in this study were in the range of 20-30%. Although the results of this quality control survey are promising, the quality of measurements has to be further optimized.

Tau protein phosphorylated at threonine 181 in CSF as a neurochemical biomarker in Alzheimer’s disease

Cerebrospinal fluid (CSF) concentrations of total Tau and Tau phosphorylated at threonine (position 181 [pTau181]) were studied with ELISA in a group of carefully selected patients with a neurochemically supported diagnosis of Alzheimer’s disease (AD, n=9; age range, 51–89 yr) and in a group of sex- and age-matched nondemented controls (n=9; age range, 52–81 yr). The concentration of both biomarkers is increased significantly in the AD group (total Tau, p<0.0008; pTau181, p<0.008). A significant correlation between CSF concentrations of both biomarkers is observed (R=0.897; p<0.001). Neither total Tau nor pTau181 correlates with age or degree of memory impairment, and only a tendency is observed between the concentrations of total Tau and Aβ42 in the CSF. Our results further confirm a possible role of pTau181 as a diagnostic tool in AD. The current literature regarding the physiological and pathological role of phosphorylated Tau proteins is reviewed, as well as the role of these proteins as promising biomarkers in the diagnosis of neurodegenerative disorders.

Amyloid β peptides in cerebrospinal fluid as profiled with surface enhanced laser desorption/ionization time-of-flight mass spectrometry: evidence of novel biomarkers in Alzheimer's disease

Abstract Background The advent of new therapeutic avenues for Alzheimers disease (AD) calls for an improved early and differential diagnosis. Methods With surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), cerebrospinal fluid from patients with AD ( n = 10) and nondemented control subjects ( n = 9) was studied. Results Molecular mass signals were observed corresponding to three novel amyloid beta (Aβ) peptides that have not previously been described, in addition to those previously known, with molecular masses of 4525.1 d, 4846.8 d, and 7755.8 d. The signal-to-noise ratios (S/NR) of Aβ(4525.1) and Aβ(7758.8+2H) were significantly decreased in AD [Aβ(4525.1): median 2.2 and 4.3 in AD and control subjects, respectively, p p p p R = .67, p Conclusions We report evidence of three novel amyloid β peptides that might play an important role in the diagnosis and pathophysiology of Alzheimers disease.

Amyloid-β 42/40 cerebrospinal fluid concentration ratio in the diagnostics of Alzheimer's disease: validation of two novel assays.

BACKGROUND The increasing role of cerebrospinal fluid (CSF) biomarkers in the early diagnosis of Alzheimers disease (AD) is reflected in recently published diagnostic and/or research criteria. A growing body of evidence suggests better diagnostic performance of the amyloid-β (Aβ)42/40 CSF concentration ratio compared to the Aβ42 concentration alone. OBJECTIVE (a) to analytically validate two novel ELISAs capable to measure Aβ1-40 and Aβ1-42 in the CSF, and (b) to compare the diagnostic accuracies of Aβ1-42 and Aβ42/40 ratio. METHODS In this study, (a) the novel Aβ1-40 and Aβ1-42 ELISAs (IBL International GmbH, Hamburg, Germany) have been analytically validated, and (b) a clinical study has been performed comparing the diagnostic performance of the CSF Aβ42/40 concentration ratio and the CSF Aβ42 concentration. RESULTS In the analytical part of the study, only marginal cross-reactivity (Aβ1-42 versus Aβ1-40) was observed; recoveries were in the range of 85-100% for the samples diluted 1 : 20-1 : 640 (Aβ1-40), and 92-104% for the samples diluted 1 : 20-1 : 320 (Aβ1-42). For Aβ1-40, the intra-assay imprecision was 2.1%, the inter-assay imprecision was 4.4%, and the inter-lot imprecision was 5.4 %. For Aβ1-42, the numbers were 3.1%, 6.2%, and 6.9%, respectively. The goodness of the fit of the average standard curves was >0.99 for both assays, and the imprecision of the optical densities in ten repetitions of the standard curves was ≤5% for all standards. In the clinical part, at the cut off value 691 pg/mL, Aβ1-42 showed sensitivity and specificity of 69.3% and 88.9%, respectively, whereas at the cut off value 0.06, the Aβ42/40 ratio showed significantly improved performance with sensitivity and specificity of 93.3% and 100%, respectively. The area under the ROC curve for Aβ42/40 (0.974) was highly significantly larger compared to Aβ1-42 concentration ROC curve (0.827, p < 0.0001). CONCLUSIONS (a) the novel Aβ1-40 and Aβ1-42 ELISA assays characterize with very good analytical performance; (b) we reconfirm that the CSF Aβ42/40 concentration ratio shows significantly better diagnostic performance compared to the CSF Aβ1-42 concentration alone.

Neurogranin and YKL-40: independent markers of synaptic degeneration and neuroinflammation in Alzheimer’s disease

IntroductionNeuroinflammation and synaptic degeneration are major neuropathological hallmarks in Alzheimer’s disease (AD). Neurogranin and YKL-40 in cerebrospinal fluid (CSF) are newly discovered markers indicating synaptic damage and microglial activation, respectively.MethodsCSF samples from 95 individuals including 39 patients with AD dementia (AD-D), 13 with mild cognitive impairment (MCI) due to AD (MCI-AD), 29 with MCI not due to AD (MCI-o) and 14 patients with non-AD dementias (non-AD-D) were analyzed for neurogranin and YKL-40.ResultsPatients with dementia or MCI due to AD showed elevated levels of CSF neurogranin (p < 0.001 for AD-D and p < 0.05 for MCI-AD) and YKL-40 (p < 0.05 for AD-D and p = 0.15 for MCI-AD) compared to mildly cognitively impaired subjects not diagnosed with AD. CSF levels of neurogranin and YKL-40 did not differ between MCI not due to AD and non-AD dementias. In AD subjects no correlation between YKL-40 and neurogranin was found. The CSF neurogranin levels correlated moderately with tau and p-tau but not with Aβ42 or the MMSE in AD samples. No relevant associations between YKL-40 and MMSE or the core AD biomarkers, Aβ42, t-tau and p-tau were found in AD subjects.ConclusionsNeurogranin and YKL-40 are promising AD biomarkers, independent of and complementary to the established core AD biomarkers, reflecting additional pathological changes in the course of AD.

Memantine inhibits ethanol-induced NMDA receptor up-regulation in rat hippocampal neurons.

The present study examined the effect of memantine, an uncompetitive NMDA receptor antagonist, on ethanol-induced NMDA receptor up-regulation. Primary glutamatergic rat hippocampal neurons were exposed to ethanol and memantine for 5 days. The ethanol-sensitive NMDA receptor subunits NR1, NR2A and NR2B were quantified by Western immunoblot analysis. Exposure to ethanol (50 mM) caused an increase in the levels of NR1 (137 +/- 11% of untreated control, P = 0.009), NR2A (128 +/- 14%, P = 0.022) and NR2B (136 +/- 19%, P = 0.012). Coincubation with memantine (10 microM) completely blocked the ethanol-induced up-regulation of NR1 (102 +/- 4%), NR2A (95 +/- 7%) and NR2B (105 +/- 13%). No effect of memantine on NR subunit expression was observable, except for NR2A, where a decrease (79 +/- 6%, P = 0.034) was noted. Neither ethanol nor memantine alone or in combination were toxic in the concentrations tested. These results may provide a molecular explanation for beneficial effects of memantine on ethanol-induced glutamatergic hyperexcitability reflected in the ethanol withdrawal syndrome and on the development of ethanol dependence.

Preanalytical Sample Handling and Sample Stability Testing for the Neurochemical Dementia Diagnostics

Preanalytical sample handling and storage procedures play an extremely important role in reliably measuring neurochemical dementia diagnostics (NDD) biomarkers: Aβ(1-40), Aβ(1-42), Tau, and pTau181. To test different handling and storage conditions, the following protocols were applied: (a) storage at room temperature for one week, (b) deep-freezing and thawing up to three cycles, (c) deep-freezing, thawing and keeping under +4°C for two days before the analysis, and (d) long-term stability of a deeply frozen sample. Between the first and the seventh day of the storage at room temperature, the percentage of the concentrations (compared to the starting concentrations) fluctuated: 104.3-105.3, 97.6-93.2, 100.6-96.8, and 97.9-90.2 for Aβ(1-40), Aβ(1-42), Tau, and pTau181, respectively. Re-freezing cycles resulted in the percentage fluctuations of the concentrations: 101.1-105.5, 95.4-99.7, 98.3-100.0, and 100.5-101.4 for Aβ(1-40), Aβ(1-42), Tau, and pTau181, respectively. Keeping previously frozen/thawed samples under +4°C for two days resulted in the percentage differences of the concentrations: +15.9, +2.2, -1.1, and -0.1 for Aβ(1-40), Aβ(1-42), Tau, and pTau181, respectively. During long-term stability, the coefficients of linear correlation (R(2)) were: Aβ(1-40), 0.007; Aβ(1-42), 0.02; Tau, 0.011; and pTau181, 0.02, and the corresponding inter-assay coefficients of variation: 13.9%, 13.9%, 11.0%, and 10.7% for Aβ(1-40), Aβ(1-42), Tau, and pTau181, respectively. We conclude that the NDD biomarkers are relatively stable when the cerebrospinal fluid sample is kept at room temperature for about four days; one or two thawing/refreezing cycles do not profoundly affect the biomarkers concentrations, however three cycles result in increased unsystematic variation. The four biomarkers seem to be stable in a sample stored deeply frozen for more than two years.

Antidepressant drugs modulate growth factors in cultured cells

BackgroundDifferent classes of antidepressant drugs are used as a treatment for depression by activating the catecholinergic system. In addition, depression has been associated with decrease of growth factors, which causes insufficient axonal sprouting and reduced neuronal damage repair. In this study, antidepressant treatments are analyzed in a cell culture system, to study the modulation of growth factors.ResultsWe quantified the transcription of several growth factors in three cell lines after application of antidepressant drugs by real time polymerase chain reaction. Antidepressant drugs counteracted against phorbolester-induced deregulation of growth factors in PMA-differentiated neuronal SY5Y cells. We also found indications in a pilot experiment that magnetic stimulation could possibly modify BDNF in the cell culture system.ConclusionThe antidepressant effects antidepressant drugs might be explained by selective modulation of growth factors, which subsequently affects neuronal plasticity.