Non-Phosphorylated Tau as a Potential Biomarker of Alzheimer's Disease: Analytical and Diagnostic Characterization.

BACKGROUND: Virtually nothing is known about a potential diagnostic role of non-phospho-epitopes of Tau (Non-P-Tau) in cerebrospinal fluid (CSF). OBJECTIVE: To establish and analytically and clinically characterize the first assay capable to measure concentrations of Non-P-Tau in human CSF. METHODS: An antibody (1G2) was developed that selectively binds to the Tau molecule non-phosphorylated at the positions T175 and T181, and was used in establishing a sandwich ELISA capable to measure Non-P-Tau in human CSF, following analytical and clinical validation of the method. RESULTS: The 1G2 antibody shows decreasing reactivity to tau peptides containing phosphorylation mainly at positions T175 and T181. Detection limit of the assay is 25 pg/ml; the coefficients of variation (CVs) of the optical densities of the repeated standard curves were between 3.6-15.9%. Median intra-assay imprecision of double measurements was 4.8%; inter-assay imprecision was in the range of 11.2% - 15.3%. Non-P-Tau concentrations are stable in the CSF samples sent to distinct laboratories under ambient temperature; inter-laboratory variation was approximately 30%. The Non-P-Tau CSF concentrations were highly significantly increased in patients with Alzheimer's disease in stage of mild cognitive impairment or dementia (AD/MCI, n = 58, 109.2±32.0 pg/mL) compared to the non-demented Controls (n = 42, 62.1±9.3 pg/mL, p < 0.001). At the cut-off of 78.3 pg/mL, the sensitivity and the specificity were 94.8% and 97.6%, respectively. CONCLUSION: For the first time, an assay is reported to reliably measure concentrations of non-phosphorylated Tau in human CSF.

Comparison of Different Matrices as Potential Quality Control Samples for Neurochemical Dementia Diagnostics.

BACKGROUND: Assay-vendor independent quality control (QC) samples for neurochemical dementia diagnostics (NDD) biomarkers are so far commercially unavailable. This requires that NDD laboratories prepare their own QC samples, for example by pooling leftover cerebrospinal fluid (CSF) samples. OBJECTIVE: To prepare and test alternative matrices for QC samples that could facilitate intra- and inter-laboratory QC of the NDD biomarkers. METHODS: Three matrices were validated in this study: (A) human pooled CSF, (B) Aß peptides spiked into human prediluted plasma, and (C) Aß peptides spiked into solution of bovine serum albumin in phosphate-buffered saline. All matrices were tested also after supplementation with an antibacterial agent (sodium azide). We analyzed short- and long-term stability of the biomarkers with ELISA and chemiluminescence (Fujirebio Europe, MSD, IBL International), and performed an inter-laboratory variability study. RESULTS: NDD biomarkers turned out to be stable in almost all samples stored at the tested conditions for up to 14 days as well as in samples stored deep-frozen (at - 80°C) for up to one year. Sodium azide did not influence biomarker stability. Inter-center variability of the samples sent at room temperature (pooled CSF, freeze-dried CSF, and four artificial matrices) was comparable to the results obtained on deep-frozen samples in other large-scale projects. CONCLUSION: Our results suggest that it is possible to replace self-made, CSF-based QC samples with large-scale volumes of QC materials prepared with artificial peptides and matrices. This would greatly facilitate intra- and inter-laboratory QC schedules for NDD measurements.

Application of multiplexing technology to the analysis of the intrathecally released immunoglobulins against B. burgdorferi antigens in neuroborreliosis.

Lyme neuroborreliosis (LNB) is an infectious disease of the nervous system caused by the tick-borne spirochete Borrelia burgdorferi. The presence of B. burgdorferi specific antibodies in cerebrospinal fluid (CSF), with evidence of intrathecal production, is the traditional diagnostic standard, although has limitations it such as low sensitivity in the very early phase. In the current study, 27 patients with possible neuroborreliosis suffered from clinically defined Bannwarth syndrome. The control group (CON) consisted of 6 patients. The analyses included function of the blood-CSF barrier (QAlb) as well as intrathecal synthesis of total IgG and IgM, (QIgG, and QIgM). Multiplexing analyses of the specific antibodies (IgG and IgM) against B.burgdorferi antigens were performed with the Microgen assay (Neuried, Germany). The ASI antibodies (Antibody Synthesis Index) specific to particular B. burgdorferi antigens (VlsE, OspC, etc.) were calculated analogously as QIgG and QIgM for separate antibody. All but one patient with NB had pathologic ASI-IgG against B. burgdorferi (median 6.3). Out of 27 NB patients, 13 had measurable ASI-IgM, and all these indices were pathologic. None of the CON subjects had pathologic ASI in either IgG or IgM class. Furthermore, NB patients showed dysfunction of the blood-CSF barrier (average QAlb in the NB and CON groups: 13.8 and 5.6, respectively, p<0.01). Twenty-one of 27 NB patients had at least one positive (>1.5) IgG-ASI against either VlsE, p100, p58, p39, p18, or OspC, and none of these patients showed positive OspA-IgG-ASI. Interestingly, the NB patient with negative IgG ASI on ELISA had the highest p100 IgG ASI on multiplexing (270.8). Among the 13 NB patients with detectable IgM-ASI on ELISA, nine showed measurable IgM-ASI against at least one antigen; however, in one of these cases, the OspC ASI was normal (0.6). In addition, one subject with non-measurable IgM ASI on ELISA had highly pathologic (19.7) index for OspC B. g. on multiplexing. The control subjects with measurable ASI-IgG on ELISA (two cases) had measurable, but normal, indices for VlsE in the IgG class also on multiplexing. None of the control subjects had measurable indices for any of the antigens in the IgM class. The simultaneous analysis of a panel of antibodies against different B. burgdorferi antigens makes multiplexing technology a very interesting supplement to the classic ELISA by providing more specific, antigen-related indices to the general, antigen-unspecific ASI. Whether this additional information proves to be diagnostically relevant will be certainly a matter of further studies.

Characterization of the postsynaptic protein neurogranin in paired cerebrospinal fluid and plasma samples from Alzheimer's disease patients and healthy controls.

INTRODUCTION: Synaptic dysfunction and degeneration are central events in Alzheimer's disease (AD) pathophysiology that are thought to occur early in disease progression. Synaptic pathology may be studied by examining protein biomarkers specific for different synaptic elements. We recently showed that the dendritic protein neurogranin (Ng), including the endogenous Ng peptide 48 to 76 (Ng48-76), is markedly increased in cerebrospinal fluid (CSF) in AD and that Ng48-76 is the dominant peptide in human brain tissue. The aim of this study was to characterize Ng in plasma and CSF using mass spectrometry and to investigate the performance of plasma Ng as an AD biomarker. METHODS: Paired plasma and CSF samples from patients with AD (n = 25) and healthy controls (n = 20) were analyzed in parallel using an immunoassay developed in-house on the Meso Scale Discovery platform and hybrid immunoaffinity-mass spectrometry (HI-MS). A second plasma material from patients with AD (n = 13) and healthy controls (n = 17) was also analyzed with HI-MS. High-resolution mass spectrometry was used for identification of endogenous plasma Ng peptides. RESULTS: Ng in human plasma is present as several endogenous peptides. Of the 16 endogenous Ng peptides identified, seven were unique for plasma and not detectable in CSF. However, Ng48-76 was not present in plasma. CSF Ng was significantly increased in AD compared with controls (P < 0.0001), whereas the plasma Ng levels were similar between the groups in both studies. Plasma and CSF Ng levels showed no correlation. CSF Ng was stable during storage at -20°C for up to 2 days, and no de novo generation of peptides were detected. CONCLUSIONS: For the first time, to our knowledge, we have identified several endogenous Ng peptides in human plasma. In agreement with previous studies, we show that CSF Ng is significantly increased in AD as compared with healthy controls. The origin of Ng in plasma and its possible use as a biomarker need to be further investigated. The results suggest that CSF Ng, in particular Ng48-76, might reflect the neurodegenerative processes within the brain, indicating a role for Ng as a potential novel clinical biomarker for synaptic function in AD.

Surface Trafficking of APP and BACE in Live Cells.

Amyloid-ß (Aß)-peptide, the major constituent of the plaques that develop during Alzheimer's disease, is generated via the cleavage of Aß precursor protein (APP) by ß-site APP-cleaving enzyme (BACE). Using live-cell imaging of APP and BACE labeled with pH-sensitive proteins, we could detect the release events of APP and BACE and their distinct kinetics. We provide kinetic evidence for the cleavage of APP by α-secretase on the cellular surface after exocytosis. Furthermore, simultaneous dual-color evanescent field illumination revealed that the two proteins are trafficked to the surface in separate compartments. Perturbing the membrane lipid composition resulted in a reduced frequency of exocytosis and affected BACE more strongly than APP. We propose that surface fusion frequency is a key factor regulating the aggregation of APP and BACE in the same membrane compartment and that this process can be modulated via pharmacological intervention.

Amyloid-ß 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 Alzheimer's disease (AD) is reflected in recently published diagnostic and/or research criteria. A growing body of evidence suggests better diagnostic performance of the amyloid-ß (Aß)42/40 CSF concentration ratio compared to the Aß42 concentration alone. OBJECTIVE: (a) to analytically validate two novel ELISAs capable to measure Aß1-40 and Aß1-42 in the CSF, and (b) to compare the diagnostic accuracies of Aß1-42 and Aß42/40 ratio. METHODS: In this study, (a) the novel Aß1-40 and Aß1-42 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 Aß42/40 concentration ratio and the CSF Aß42 concentration. RESULTS: In the analytical part of the study, only marginal cross-reactivity (Aß1-42 versus Aß1-40) was observed; recoveries were in the range of 85-100% for the samples diluted 1 : 20-1 : 640 (Aß1-40), and 92-104% for the samples diluted 1 : 20-1 : 320 (Aß1-42). For Aß1-40, the intra-assay imprecision was 2.1%, the inter-assay imprecision was 4.4%, and the inter-lot imprecision was 5.4 %. For Aß1-42, 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, Aß1-42 showed sensitivity and specificity of 69.3% and 88.9%, respectively, whereas at the cut off value 0.06, the Aß42/40 ratio showed significantly improved performance with sensitivity and specificity of 93.3% and 100%, respectively. The area under the ROC curve for Aß42/40 (0.974) was highly significantly larger compared to Aß1-42 concentration ROC curve (0.827, p < 0.0001). CONCLUSIONS: (a) the novel Aß1-40 and Aß1-42 ELISA assays characterize with very good analytical performance; (b) we reconfirm that the CSF Aß42/40 concentration ratio shows significantly better diagnostic performance compared to the CSF Aß1-42 concentration alone.

Ventricular and lumbar cerebrospinal fluid concentrations of Alzheimer's disease biomarkers in patients with normal pressure hydrocephalus and posttraumatic hydrocephalus.

BACKGROUND: Little information is available on the rostro-caudal concentration gradient of Alzheimer's disease (AD) biomarkers. OBJECTIVE: We studied the concentrations of amyloid-ß (Aß) peptides 1-42 and 1-40 as well as the Tau and pTau proteins in simultaneously collected ventricular and lumbar cerebrospinal fluid (CSF) samples. METHODS: The samples were simultaneously collected from the ventricle and the lumbar spinal canal in two groups of patients: 10 subjects being treated for normal pressure hydrocephalus (NPH) by the placement of a ventriculo-peritoneal shunt and 5 patients treated simultaneously with an external ventricular drain and a lumbar CSF drain due to posttraumatic hydrocephalus (PTH). RESULTS: The ventricular-lumbar (V/L) concentration ratio for Aß1-40 was 0.81 in NPH patients and 0.71 in PTH patients. The V/L-ratio for Aß1-42 was 0.84 in NPH, reflecting significantly higher concentrations in lumbar CSF than in ventricular CSF, and 1.02 in PTH patients. The V/L-ratios for Tau and pTau differed significantly depending on the diagnostic group: the median V/L-ratio for Tau was 6.83 in NPH patients but only 0.97 in PTH patients. The median V/L-ratio for pTau was 2.36 in NPH patients and 0.91 in PTH patients. CONCLUSIONS: We conclude that the rostro-caudal concentration gradient for brain-derived proteins (Tau and pTau in this study) depends on the diagnosis and clinical status of the patient, which were largely neglected in the previously postulated models.

Plasma concentrations of the amyloid-ß peptides in young volunteers: the influence of the APOE genotype.

Changes in the concentrations of amyloid-ß (Aß) in the body fluids are the earliest alterations observed in Alzheimer's disease (AD), however, there is a lack of data about how early these alterations occur, before the onset of the clinical symptoms. APOE genotype is the most recognized genetic risk/protective factor of AD, meaning that a group of non-demented persons carrying ε4 allele is enriched in the subjects who will develop AD, compared to the group of non-carriers. Therefore, we studied the plasma concentrations of Aß peptides (Aß1-42, Aß1-40, Aßx-42, and Aßx-40), and the APOE genotype in 173 young volunteers (average age, 28 ± 7.6 years) without memory deficits, in order to see whether the non-demented group of subjects at risk already characterize with Aß changes three-to-four decades before the age at which dementia usually occurs. We did not find statistically significant differences among the groups of ε4 carriers, ε3 homozygotes, and ε2 carriers. We conclude that the APOE genotype does not influence the metabolism of the Aß peptides in young persons without memory deficits.

Neuroprotein dynamics in the cerebrospinal fluid: intraindividual concomitant ventricular and lumbar measurements.

OBJECTIVE: The measurement of neuromarker/neuroproteins in the cerebrospinal fluid (CSF) is gaining increased popularity. However, insufficient information is available on the rostrocaudal distribution of neuroproteins in the CSF to guarantee an appropriate interpretation of ventricular versus lumbar concentrations. METHODS: In 10 patients treated with both an external ventricular and a lumbar CSF drain, we collected concomitant CSF samples. We measured CSF concentrations of the glial S100B protein, the neuron-specific enolase (Cobas e411®; Roche Diagnostics), the leptomeningeal ß-trace protein (BN Pro Spec®; Dade Behring/Siemens), and the blood-derived albumin (Immage; Beckman Coulter). Statistical analysis was performed with a paired Wilcoxon signed ranks test. RESULTS: In patients with a free CSF circulation without any recent neurosurgical procedure, S100B and neuron-specific enolase concentrations did not differ between the ventricular and lumbar CSF while ß-trace and albumin levels were significantly higher in the lumbar than in the ventricular CSF (p=0.008 and p=0.005). Following posterior fossa tumor surgery, all proteins accumulate in the lumbar CSF. CONCLUSION: For brain-derived proteins, we could not confirm a rostrocaudal CSF gradient while lepto-meningeal and blood-derived proteins accumulate in the lumbar CSF. We conclude that for the interpretation of protein CSF concentrations, the source of the sample is of crucial importance.

The influence of insulin infusion on the metabolism of amyloid ß peptides in plasma.

BACKGROUND: Accumulating body of evidence suggests pathophysiologic links between Alzheimer's disease and diabetes mellitus (DM). For example, the two crucial peptides playing a role in both degenerative disorders, amyloid ß (Aß) and insulin, are metabolized by the same enzyme, insulin degrading enzyme. Euglycemic hyperinsulinemic clamp is a method of estimating insulin sensitivity, based on the assumption that during steady-state hyperinsulinemic euglycemia, glucose infusion rate equals tissue glucose uptake, that is, the higher the glucose infusion rate, the higher the insulin sensitivity. OBJECTIVE: The aim of this study was to analyze the influence of insulin on the plasma concentrations of Aß peptides. METHODS: Blood samples were collected from 20 healthy young male volunteers before insulin infusion (clamp) and then at 120 and 360 minutes. In the second protocol, insulin was accompanied by Intralipid, which is mainly a mixture of triacylglycerols, and heparin, given as an activator of lipoprotein lipase, inducing insulin resistance. Analyses of plasma Aß1-42, Aßx-42, Aß1-40, and Aßx-40 were performed with multiplexing technology. Furthermore, concentrations of the Aß peptides in healthy persons were compared with those in 16 type 1 DM patients receiving chronic insulin therapy. RESULTS: When applied alone (i.e., without Intralipid), insulin infusion increased concentrations of Aß42 (full length and N-terminally shortened) but not of Aß40. When combined with Intralipid, infusion of insulin resulted in increased concentrations of all peptides (nonsignificant tendency in case of Aßx-40). We did not observe differences between Aß peptide concentrations in healthy subjects and those in type 1 DM patients. CONCLUSION: Infusion of insulin in nonphysiologic high doses increases plasma concentrations of Aß peptides; in case of Aß40, only when applied together with Intralipid, which perhaps might be explained by hypothetical shift of insulin degrading enzyme activity from degradation of Aß peptides to the degradation of insulin.

Multiplexing analysis of the polyspecific intrathecal immune response in multiple sclerosis.

Intrathecal synthesis of the antibodies specific to neurotrofic viruses: measles (M), rubella (R), Varicella-Zoster (Z), and/or H. simplex (H), known as "MRZH-reaction" plays important diagnostic role in multiple sclerosis (MS). Whereas the analysis of the oligoclonal IgG bands provides high sensitivity, the MRZH-reaction shows high specificity, and hence these methods complement each other. For the first time we applied multiplexing bead-based technology to simultaneously analyze cerebrospinal fluid (CSF) and serum concentrations of antibodies against these viruses, and to calculate the antibody specific indices (ASI's). The method shows reasonable precision: intra-assay, 2.9-6.7%, and inter-assay, 2.0-3.2%. The results are comparable with these obtained with other methods (ELISAs), including two runs of the certified external quality control schemes. Eighty-one percent of the MS cases (n=27) and none of the sex- and age-matched controls (n=14), except one subject with "borderline" anti-measles ASI of 1.5, showed intrathecal synthesis of IgG against at least one of the viruses discussed. The ratios of the MRZH-positive cases in the MS group were: 12/22 for M, 12/19 for R, 13/26 for Z, and 7/26 for H. We conclude that the multiplexing technology can be applied as a tool to study the intrathecal immune response in the diagnosis of MS.

Cerebrospinal fluid soluble amyloid-ß protein precursor as a potential novel biomarkers of Alzheimer's disease.

In this report, we confirm our previous findings of increased concentrations of soluble amyloid-ß protein precursor (sAßPP) in cerebrospinal fluid (CSF) of patients with Alzheimer's disease (AD) and mild cognitive impairment (MCI) in a large cohort of patients (n = 314), not overlapping with those of our previous study, and we extend our observations by including a control group of participants with normal cognition. In addition, we investigate the effects of age, the APOEε4 genotype, and the blood-CSF barrier function on the concentrations of sAßPPα and sAßPPß. The study participants were categorized according to clinical-neuropsychological criteria, supported by CSF neurochemical dementia diagnostics (NDD) analyses. sAßPPα concentrations in the AD group (132.0 ± 44.8) were significantly higher than in the control group (105.3 ± 37.3, p < 0.0005) but did not differ from the MCI-AD group (138.5 ± 39.5, p = 0.91). The MCI-AD group differed significantly from the MCI-O (97.3 ± 34.3, p < 0.05) group. There was no difference between the control and the MCI-O groups (p = 0.94). Similarly, sAßPPß concentrations in the AD group (160.2 ± 54.3) were significantly higher than in the control group (129.9 ± 44.6, p < 0.005) but did not differ from the MCI-AD group (184.0 ± 56.4, p = 0.20). The MCI-AD group differed significantly from the MCI-O (127.8 ± 46.2, p < 0.05) group. There was no difference between the control and the MCI-O groups (p > 0.99). We observed highly significant correlation of the two sAßPP forms. Age and the CSF-serum albumin ratio were significant albeit weak predictors of the sAßPPα and sAßPPß concentrations, while carrying the APOEε4 allele did not influenced the levels of the sAßPP forms. Taken together, the results strongly suggest that CSF sAßPP concentrations may be considered as an extension of already available NDD tools.

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: Aß(1-40), Aß(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 Aß(1-40), Aß(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 Aß(1-40), Aß(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 Aß(1-40), Aß(1-42), Tau, and pTau181, respectively. During long-term stability, the coefficients of linear correlation (R(2)) were: Aß(1-40), 0.007; Aß(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 Aß(1-40), Aß(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.