Automated Capillary Electrophoresis Immunoblot for the Detection of Alpha-Synuclein in Mouse Tissue.

Background: Alpha-synuclein (aSyn) is a key player in neurodegenerative diseases such as Parkinson's disease (PD), dementia with Lewy bodies, or multiple system atrophy. aSyn is expressed throughout the brain, and can also be detected in various peripheral tissues. In fact, initial symptoms of PD are non-motoric and include autonomic dysfunction, suggesting that the periphery might play an important role in early development of the disease. aSyn is expressed at relatively low levels in non-central tissues, which brings challenges for its detection and quantification in different tissues. Objective: Our goal was to assess the sensitivity of aSyn detection in central and peripheral mouse tissues through capillary electrophoresis (CE) immunoblot, considering the traditional SDS-PAGE immunoblot as the current standard. Methods: Tissues from central and non-central origin from wild type mice were extracted, and included midbrain, inner ear, and esophagus/stomach. aSyn detection was assessed through immunoblotting using Simple Western size-based CE and SDS-PAGE. Results: CE immunoblots show a consistent detection of aSyn in central and peripheral tissues. Through SDS-PAGE, immunoblots revealed a reliable signal corresponding to aSyn, particularly following membrane fixation. Conclusion: Our results suggest a reliable detection of aSyn in central and peripheral tissues using the CE Simple Western immunoblot system. These observations can serve as preliminary datasets when aiming to formally compare CE with SDS-PAGE, as well as for further characterization of aSyn using this technique.

Diagnostic performance of automated plasma amyloid-ß assays combined with pre-analytical immunoprecipitation.

BACKGROUND: Measurements of the amyloid-ß (Aß) 42/40 ratio in blood plasma may support the early diagnosis of Alzheimer's disease and aid in the selection of suitable participants in clinical trials. Here, we compared the diagnostic performance of fully automated prototype plasma Aß42/40 assays with and without pre-analytical sample workup by immunoprecipitation. METHODS: A pre-selected clinical sample comprising 42 subjects with normal and 38 subjects with low cerebrospinal fluid (CSF) Aß42/40 ratios was studied. The plasma Aß42/40 ratios were determined with fully automated prototype Elecsys® immunoassays (Roche Diagnostics GmbH, Penzberg, Germany) by direct measurements in EDTA plasma or after pre-analytical Aß immunoprecipitation. The diagnostic performance for the detection of abnormal CSF Aß42/40 was analyzed by receiver operating characteristic (ROC) analysis. In an additional post hoc analysis, a biomarker-supported clinical diagnosis was used as a second endpoint. RESULTS: Pre-analytical immunoprecipitation resulted in a significant increase in the area under the ROC curve (AUC) from 0.73 to 0.88 (p = 0.01547) for identifying subjects with abnormal CSF Aß42/40. A similar improvement in the diagnostic performance by pre-analytical immunoprecipitation was also observed when a biomarker-supported clinical diagnosis was used as a second endpoint (AUC increase from 0.77 to 0.92, p = 0.01576). CONCLUSIONS: Our preliminary observations indicate that pre-analytical Aß immunoprecipitation can improve the diagnostic performance of plasma Aß assays for detecting brain amyloid pathology. The findings may aid in the further development of blood-based immunoassays for Alzheimer's disease ultimately suitable for screening and routine use.

Development and Technical Validation of an Immunoassay for the Detection of APP669-711 (Aß-3-40) in Biological Samples.

The ratio of amyloid precursor protein (APP)669-711 (Aß-3-40)/Aß1-42 in blood plasma was reported to represent a novel Alzheimer's disease biomarker. Here, we describe the characterization of two antibodies against the N-terminus of Aß-3-x and the development and "fit-for-purpose" technical validation of a sandwich immunoassay for the measurement of Aß-3-40. Antibody selectivity was assessed by capillary isoelectric focusing immunoassay, Western blot analysis, and immunohistochemistry. The analytical validation addressed assay range, repeatability, specificity, between-run variability, impact of pre-analytical sample handling procedures, assay interference, and analytical spike recoveries. Blood plasma was analyzed after Aß immunoprecipitation by a two-step immunoassay procedure. Both monoclonal antibodies detected Aß-3-40 with no appreciable cross reactivity with Aß1-40 or N-terminally truncated Aß variants. However, the amyloid precursor protein was also recognized. The immunoassay showed high selectivity for Aß-3-40 with a quantitative assay range of 22 pg/mL-7.5 ng/mL. Acceptable intermediate imprecision of the complete two-step immunoassay was reached after normalization. In a small clinical sample, the measured Aß42/Aß-3-40 and Aß42/Aß40 ratios were lower in patients with dementia of the Alzheimer's type than in other dementias. In summary, the methodological groundwork for further optimization and future studies addressing the Aß42/Aß-3-40 ratio as a novel biomarker candidate for Alzheimer's disease has been set.

A two-step immunoassay for the simultaneous assessment of Aß38, Aß40 and Aß42 in human blood plasma supports the Aß42/Aß40 ratio as a promising biomarker candidate of Alzheimer's disease.

BACKGROUND: The quantification of amyloid-beta (Aß) peptides in blood plasma as potential biomarkers of Alzheimer's disease (AD) is hampered by very low Aß concentrations and the presence of matrix components that may interfere with the measurements. METHODS: We developed a two-step immunoassay for the simultaneous measurement of the relative levels of Aß38, Aß40 and Aß42 in human EDTA plasma. The assay was employed for the study of 23 patients with dementia of the Alzheimer's type (AD-D) and 17 patients with dementia due to other reasons (OD). We examined relationships with the clinical diagnosis, cerebral Aß load as quantified by amyloid-positron emission tomography, apolipoprotein E genotype, Aß levels and Tau protein in cerebrospinal fluid. RESULTS: Preconcentration of plasma Aß peptides by immunoprecipitation substantially facilitated their immunological measurements. The Aß42/Aß40 and Aß42/Aß38 ratios were statistically significantly lower in the AD-D patients than in the OD group. The areas under the receiver operating characteristic curves reached 0.87 for the Aß42/Aß40 ratio and 0.80 for the Aß42/Aß38 ratio. CONCLUSIONS: The measurement of plasma Aß peptides with an immunological assay can be improved by preconcentration via immunoprecipitation with an antibody against the Aß amino-terminus and elution of the captured peptides by heating in a mild detergent-containing buffer. Our findings support the Aß42/Aß40 ratio in blood plasma as a promising AD biomarker candidate which correlates significantly with the validated core biomarkers of AD. Further studies will be needed for technical advancement of the assay and validation of the biomarker findings.

Micro-droplet arrays for micro-compartmentalization using an air/water interface.

Here we present a water-in-air droplet platform for micro-compartmentalization for single molecule guided synthesis and analysis consisting of a flow-system hosting dense arrays of aqueous microdroplets on a glass surface surrounded by air. The droplets are formed in a few seconds by passing a waterfront over the array of hydrophilic spots surrounded by a hydrophobic coating, thus forming a micro-droplet array (MDA). The droplet volumes are tunable from approximately 50 femtoliter to 20 picoliter by adjusting the size of the hydrophilic spots. MDAs consisting of femtoliter volume droplets were stable for more than 24 hours in air at 37 °C in a reversibly sealed flow-system, thus allowing us to perform assays that require long incubations in the droplets. Using differently fluorescing liquids, it was further shown that droplets can be reformed on the same MDA several times by passing a new liquid plug over the surface, and that fluorescence from one reaction can be washed away with little to no carry-over, hence allowing for multistep reactions to be carried out on the system. The MDA created by an air/water interface supported digital immunoassays as was demonstrated by measuring the Aß42 peptide in cerebrospinal fluid of Alzheimers patients and control patients. To demonstrate a two step droplet assay, first, histidine tagged peptides were expressed in the droplets and bound to the droplet-enclosed surface. Subsequently, the his-tagged peptides were detected using enzyme-conjugated antibodies in a second droplet generation step. As such, the chip demonstrates features necessary for library preparations for high throughput screening applications.

Validation of a Chemiluminescence Immunoassay for Measuring Amyloid-ß in Human Blood Plasma.

The technical performance of immunological assays and their suitability for the intended use should be carefully validated before implementation in research, clinical studies or routine. We describe here the evaluation of a sandwich electrochemiluminescence immunoassay for measuring total Amyloid-ß levels in human blood plasma as an example of a laboratory protocol for a partial "fit for purpose" assay performance validation. We tested two different assay protocols and addressed impact of sample dilution, parallelism, intra- and inter-assay variance, lower limit of quantification, lower limit of detection, and analytical spike recoveries.

Reproducibility of Alzheimer's Disease Cerebrospinal Fluid-Biomarker Measurements under Clinical Routine Conditions.

Analysis of cerebrospinal fluid (CSF) is one of the key tools for the state-of-the-art differential diagnosis of dementias. Dementia due to Alzheimer's disease (AD) is characterized by elevated CSF levels of total Tau (tTau) and phospho-181-Tau (pTau) and low CSF amyloid-ß42 (Aß42). Discrepancies in the laboratory analysis of human materials are well known and much effort has been put into harmonization procedures. In this study, we measured CSF biomarkers of more than 100 patients obtained under clinical routine conditions in two different clinical laboratories. The CSF biomarker levels obtained from the two different sites were significantly correlated: R2 = 0.7129 (tTau, p < 0.001), 0.7914 (pTau, p < 0.001), 0.5078 (Aß42, p < 0.001), 0.5739 (Aß40, p < 0.001), and 0.4308 (Aß42/40, p < 0.001). However, the diagnostic classifications of the Aß42, tTau, and pTau levels of identical subjects into normal versus pathological range made by the two different sites showed substantial discrepancies (31.5%, 29.6%, and 25.0% discordant cases, respectively). Applying Aß42/40, instead of CSF Aß42 alone, lead to a reduction of the discordant cases to 16.8%. Our findings suggest that CSF Aß42/40 can outperform Aß42 as a biomarker for AD neuropathology, not only under well-controlled study conditions but also in real life clinical routine. Thus, we recommend the inclusion of Aß42/40 as a CSF biomarker in the diagnostic procedure.

N-truncated Aß4-x peptides in sporadic Alzheimer's disease cases and transgenic Alzheimer mouse models.

BACKGROUND: The deposition of neurotoxic amyloid-ß (Aß) peptides in plaques in the brain parenchyma and in cerebral blood vessels is considered to be a key event in Alzheimer's disease (AD) pathogenesis. Although the presence and impact of full-length Aß peptides such as Aß1-40 and Aß1-42 have been analyzed extensively, the deposition of N-terminally truncated Aß peptide species has received much less attention, largely because of the lack of specific antibodies. METHODS: This paper describes the generation and characterization of novel antibodies selective for Aß4-x peptides and provides immunohistochemical evidence of Aß4-x in the human brain and its distribution in the APP/PS1KI and 5XFAD transgenic mouse models. RESULTS: The Aß4-x staining pattern was restricted mainly to amyloid plaque cores and cerebral amyloid angiopathy in AD and Down syndrome cases and in both AD mouse models. In contrast, diffuse amyloid deposits were largely negative for Aß4-x immunoreactivity. No overt intraneuronal staining was observed. CONCLUSIONS: The findings of this study are consistent with previous reports demonstrating a high aggregation propensity of Aß4-x peptides and suggest an important role of these N-truncated Aß species in the process of amyloidogenesis and plaque core formation.

Validation of a Commercial Chemiluminescence Immunoassay for the Simultaneous Measurement of Three Different Amyloid-ß Peptides in Human Cerebrospinal Fluid and Application to a Clinical Cohort.

A comprehensive assay validation campaign of a commercially available chemiluminescence multiplex immunoassay for the simultaneous measurement of the amyloid-ß peptides Aß38, Aß40, and Aß42 in human cerebrospinal fluid (CSF) is presented. The assay quality parameters we addressed included impact of sample dilution, parallelism, lower limits of detection, lower limits of quantification, intra- and inter-assay repeatability, analytical spike recoveries, and between laboratory reproducibility of the measurements. The assay performed well in our hands and fulfilled a number of predefined acceptance criteria. The CSF levels of Aß40 and Aß42 determined in a clinical cohort (n = 203) were statistically significantly correlated with available ELISA data of Aß1-40 (n = 158) and Aß1-42 (n = 179) from a different laboratory. However, Bland-Altman method comparison indicated systematic differences between the assays. The data presented here furthermore indicate that the CSF concentration of Aß40 can surrogate total CSF Aß and support the hypothesis that the Aß42/Aß40 ratio outperforms CSF Aß42 alone as a biomarker for Alzheimer's disease due to a normalization to total Aß levels.

Deposition of C-terminally truncated Aß species Aß37 and Aß39 in Alzheimer's disease and transgenic mouse models.

In Alzheimer's disease (AD) a variety of amyloid ß-peptides (Aß) are deposited in the form of extracellular diffuse and neuritic plaques (NP), as well as within the vasculature. The generation of Aß from its precursor, the amyloid precursor protein (APP), is a highly complex procedure that involves subsequent proteolysis of APP by ß- and γ-secretases. Brain accumulation of Aß due to impaired Aß degradation and/or altered ratios between the different Aß species produced is believed to play a pivotal role in AD pathogenesis. While the presence of Aß40 and Aß42 in vascular and parenchymal amyloid have been subject of extensive studies, the deposition of carboxyterminal truncated Aß peptides in AD has not received comparable attention. In the current study, we for the first time demonstrate the immunohistochemical localization of Aß37 and Aß39 in human sporadic AD (SAD). Our study further included the analysis of familial AD (FAD) cases carrying the APP mutations KM670/671NL, E693G and I716F, as well as a case of the PSEN1 ΔExon9 mutation. Aß37 and Aß39 were found to be widely distributed within the vasculature in the brains of the majority of studied SAD and FAD cases, the latter also presenting considerable amounts of Aß37 containing NPs. In addition, both peptides were found to be present in extracellular plaques but only scarce within the vasculature in brains of a variety of transgenic AD mouse models. Taken together, our study indicates the importance of C-terminally truncated Aß in sporadic and familial AD and raises questions about how these species are generated and regulated.

Beta-amyloid peptide variants in brains and cerebrospinal fluid from amyloid precursor protein (APP) transgenic mice: comparison with human Alzheimer amyloid.

In this study, we report a detailed analysis of the different variants of amyloid-ß (Aß) peptides in the brains and the cerebrospinal fluid from APP23 transgenic mice, expressing amyloid precursor protein with the Swedish familial Alzheimer disease mutation, at different ages. Using one- and two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry, we identified the Aß peptides Aß(1-40), -(1-42), -(1-39), -(1-38), -(1-37), -(2-40), and -(3-40) as well as minor amounts of pyroglutamate-modified Aß (Aß(N3pE)) and endogenous murine Aß in brains from 24-month-old mice. Chemical modifications of the N-terminal amino group of Aß were identified that had clearly been introduced during standard experimental procedures. To address this issue, we additionally applied amyloid extraction in ultrapure water. Clear differences between APP23 mice and Alzheimer disease (AD) brain samples were observed in terms of the relative abundance of specific variants of Aß peptides, such as Aß(N3pE), Aß(1-42), and N-terminally truncated Aß(2/3-42). These differences to human AD amyloid were also noticed in a related mouse line transgenic for human wild type amyloid precursor protein. Taken together, our findings suggest different underlying molecular mechanisms driving the amyloid deposition in transgenic mice and AD patients.

A method to prevent cross contamination during 2-DE by ß-amyloid peptides.

A method for the efficient decontamination of aluminium oxide ceramic 2-DE focusing trays from ß-amyloid peptides (Aß) is reported. As these contaminations were resistant to the standard cleaning procedures, additional harsh cleaning steps were necessary for their efficient removal. Our observations suggest that specific surface properties affect the degree of adsorption of the Aß-peptides. "Surface catalysed amyloid aggregation" in the aluminium oxide ceramic trays is proposed as a possible underlying mechanism for the occurrence of proteinase K-resistant forms of Aß.

Inhibition of PHF-like tau hyperphosphorylation in SH-SY5Y cells and rat brain slices by K252a.

Abnormal hyperphosphorylation of tau is believed to constitute a critical biochemical event in the process of neurofibrillary degeneration of Alzheimer's disease. We have developed a cellular model where apparently authentic PHF-like tau hyperphosphorylation is induced by okadaic acid. To gain deeper insight into the complex mechanisms of this pathological process we tested a variety of kinase inhibitors in this model. We found that K252a is differentiated from staurosporine by its inhibition of ERK2: both compounds are structurally related microbial metabolites generally believed to have only moderate kinase selectivity. However, since ERK2 inhibitors are exceedingly rare, we used this differential inhibitory property of K252a to demonstrate the involvement of ERK2 in PHF-type tau hyperphosphorylation. K252a was uniquely able to completely suppress the okadaic acid-induced tau hyperphosphorylation in SH-SY5Y cells and rat brain slices by way of including ERK2 in its inhibitory spectrum, and to conserve the normal binding of tau to tubulin. GSK3 inhibitors partially affected the normal state of tau phosphorylation in SH-SY5Y cells, but had no impact on okadaic acid-induced tau hyperhosphorylation. As K252a is the first molecule identified capable of preventing the spectrum of PHF-like tau hyperphosphorylation markers, it may represent a conceptual starting point for therapeutic development of suitable spectrum kinase inhibitors.

An inhibitor of tau hyperphosphorylation prevents severe motor impairments in tau transgenic mice.

An orally bioavailable and blood-brain barrier penetrating analog of the kinase inhibitor K252a was able to prevent the typical motor deficits in the tau (P301L) transgenic mouse model (JNPL3) and markedly reduce soluble aggregated hyperphosphorylated tau. However, neurofibrillary tangle counts were not reduced in the successfully treated cohort, suggesting that the main cytotoxic effects of tau are not exerted by neurofibrillary tangles but by lower molecular mass aggregates of tau. Our findings strongly suggest that abnormal tau hyperphosphorylation plays a critical role in the development of tauopathy and suggest a previously undescribed treatment strategy for neurodegenerative diseases involving tau pathology.