Lenka Hromadkova

Identification and characterization of natural antibodies against tau protein in an intravenous immunoglobulin product

The latest therapeutic approaches to Alzheimer disease are using intravenous immunoglobulin (IVIG) products. Therefore, the detailed characterization of target-specific antibodies naturally occurring in IVIG products is beneficial. We have focused on characterization of antibodies isolated against tau protein, a biomarker of Alzheimers disease, from Flebogamma IVIG product. The analysis of IgG subclass distribution indicated skewing toward IgG3 in anti-tau-enriched IgG fraction. The evaluation of their reactivity and avidity with several recombinant tau forms was performed by ELISA and blotting techniques. Truncated non-phosphorylated tau protein (amino acids 155-421) demonstrated the highest reactivity and avidity index. We provide the first detailed insight into the reactivity of isolated natural antibodies against tau protein.

Monoamine oxidase B is elevated in Alzheimer disease neurons, is associated with γ-secretase and regulates neuronal amyloid β-peptide levels

BackgroundIncreased levels of the pathogenic amyloid β-peptide (Aβ), released from its precursor by the transmembrane protease γ-secretase, are found in Alzheimer disease (AD) brains. Interestingly, monoamine oxidase B (MAO-B) activity is also increased in AD brain, but its role in AD pathogenesis is not known. Recent neuroimaging studies have shown that the increased MAO-B expression in AD brain starts several years before the onset of the disease. Here, we show a potential connection between MAO-B, γ-secretase and Aβ in neurons.MethodsMAO-B immunohistochemistry was performed on postmortem human brain. Affinity purification of γ-secretase followed by mass spectrometry was used for unbiased identification of γ-secretase-associated proteins. The association of MAO-B with γ-secretase was studied by coimmunoprecipitation from brain homogenate, and by in-situ proximity ligation assay (PLA) in neurons as well as mouse and human brain sections. The effect of MAO-B on Aβ production and Notch processing in cell cultures was analyzed by siRNA silencing or overexpression experiments followed by ELISA, western blot or FRET analysis. Methodology for measuring relative intraneuronal MAO-B and Aβ42 levels in single cells was developed by combining immunocytochemistry and confocal microscopy with quantitative image analysis.ResultsImmunohistochemistry revealed MAO-B staining in neurons in the frontal cortex, hippocampus CA1 and entorhinal cortex in postmortem human brain. Interestingly, the neuronal staining intensity was higher in AD brain than in control brain in these regions. Mass spectrometric data from affinity purified γ-secretase suggested that MAO-B is a γ-secretase-associated protein, which was confirmed by immunoprecipitation and PLA, and a neuronal location of the interaction was shown. Strikingly, intraneuronal Aβ42 levels correlated with MAO-B levels, and siRNA silencing of MAO-B resulted in significantly reduced levels of intraneuronal Aβ42. Furthermore, overexpression of MAO-B enhanced Aβ production.ConclusionsThis study shows that MAO-B levels are increased not only in astrocytes but also in pyramidal neurons in AD brain. The study also suggests that MAO-B regulates Aβ production in neurons via γ-secretase and thereby provides a key to understanding the relationship between MAO-B and AD pathogenesis. Potentially, the γ-secretase/MAO-B association may be a target for reducing Aβ levels using protein–protein interaction breakers.

Characterization of isolated tau-reactive antibodies from the IVIG product, plasma of patients with Alzheimer's disease and cognitively normal individuals

The presence of natural tau-reactive antibodies was reported in human blood. In this study, we isolated and characterized natural tau-reactive antibodies occurring in IVIG product Flebogamma, plasma of patients with Alzheimers disease (AD) and older cognitively normal persons (controls). Using blotting immunoassays and ELISA, we showed reactivity of antibodies obtained from IVIG and controls against a recombinant fragment of tau (155-421aa) and aggregates present in brains of AD patients. In contrast, antibodies isolated from plasma of AD patients reacted mainly with the recombinant full-length tau form and tau monomeric forms in brain tissue.

Difficulties associated with the structural analysis of proteins susceptible to form aggregates: The case of Tau protein as a biomarker of Alzheimer's disease

Mass spectrometry coupled with bioaffinity separation techniques is considered a powerful tool for studying protein interactions. This work is focused on epitope analysis of tau protein, which contains two VQIXXK aggregation motifs regarded as crucial elements in the formation of paired helical filaments, the main pathological characteristics of Alzheimers disease. To identify major immunogenic structures, the epitope extraction technique utilizing protein fragmentation and magnetic microparticles functionalized with specific antibodies was applied. However, the natural adhesiveness of some newly generated peptide fragments devalued the experimental results. Beside presumed peptide fragment specific to applied monoclonal anti-tau antibodies, the epitope extraction repeatedly revealed inter alia tryptic fragment 299-HVPGGGSVQIVYKPVDLSK-317 containing the fibril-forming motif 306-VQIVYK-311. The tryptic fragment pro-aggregation and hydrophobic properties that might contribute to adsorption phenomenon were examined by Thioflavin S and reversed-phase chromatography. Several conventional approaches to reduce the non-specific fragment sorption onto the magnetic particle surface were performed, however with no effect. To avoid methodological complications, we introduced an innovative approach based on altered proteolytic digestion. Simultaneous fragmentation of tau protein by two immobilized proteases differing in the cleavage specificity (TPCK-trypsin and α-chymotrypsin) led to the disruption of motif responsible for undesirable adhesiveness and enabled us to obtain undistorted structural data.

INTRANEURONAL AMYLOID β-PEPTIDE LEVELS ARE AFFECTED BY MONOAMINE OXIDASE B

Background: The role of vascular disease in Alzheimer’s disease (AD) is still debated. White matter hypointensity (WMH) volume is an imaging measure of vascular burden. Using MRI and Positron Emission Tomography (PET) we examined the voxel-level relationships betweenWMHand 3 key processes inAD: amyloid deposition, tau aggregation and microglial activation. Methods: 15 participants with a clinical diagnosis of AD had T1-weighted MRI, F-flutemetamol, F-AV1451 and C-PBR28 PET scans. 18 healthy controls also had F-flutemetamol and C-PBR28 scans, and 8 of these also had F-AV1451 PET. Z-score maps for each tracer (80-100 minute ratio image for F-AV1451, 90-120 minute ratio image for F-flutemetamol and logan Vd parametric map for C-PBR28 ) were calculated for the AD subjects compared to the controls for each tracer.WMHvolumes from theMRIwere calculated from Freesurfer software. Multiple regression in SPM8 was used to calculate voxel wise correlations between WMH volume and tracer uptake. A pvalue of p<0.05 was considered significant. Results:Amyloid deposition: there were correlations between WMH volume and amyloid deposition in the 1) frontal lobe (right medial and anterior orbital gyri, left posterior orbital gyrus, and right superior frontal gyri) 2) parietal lobe (left superior parietal gyrus) and 3) corpus callosum. Tau aggregation: there were correlations between WMH burden and tau aggregation in the left and right posterior temporal lobe Microglial activation :there were correlations between WMH volume and microglial activation in the 1) frontal lobe (right superior/middle frontal gyrus, right subgenual frontal cortex, right medial orbital gyrus) 2) parietal lobe (postcentral / precentral gyrus, left inferolateral part of parietal lobe and 3) occipital lobe (left lateral part of occipital lobe). Conclusions:White matter hypointensity volume correlates at voxel-level with amyloid deposition, tau aggregation and microglial activation. These findings emphasise the complex disease pathogenesis occurring in AD, and the need for a multi-targeted treatment.

Immunoaffinity purification and characterization of specific antibodies against recombinant tau protein from intravenous immunoglobulin product

Institute of Clinical Neuroimmunology, Klinikum Grosshadern, Ludwig Maximilians University, Munich, Germany; Neuroproteomics, Klinikum rechts der Isar, Technical University, Munich, Germany; German Center for Neurodegenerative Diseases (DZNE), Munich, Germany; Institute for Advanced Study, Technical University, Munich, Germany; Research Unit Protein Science, Helmholtz ZentrumMunchen, Deutsches Forschungszentrum fur Gesundheit und Umwelt (GmbH), Neuherberg, Germany; Department of Neuroimmunology, Max Planck Institute of Neurobiology, Martinsried, Germany; Division of Clinical Neuroscience, Karolinska University Hospital, Stockholm, Sweden; Department of Medicine III, Ludwig Maximilians University, Munich, Germany; Department of Neurology, Ludwig Maximilians University, Munich, Germany; Department of Rheumatology and Clinical Immunology, Charite — University Medicine, Berlin, Germany; Department of Cancer Biology, Mayo Clinic Florida, Jacksonville, FL, USA