Throughout development, neuronal progenitors undergo complex transformation into polarized nerve cells, warranting the directional flow of information in the neural grid. The majority of neuronal polarization studies have been carried out on rodent-derived precursor cells, programmed to develop into neurons. Unlike rodent neuronal cells, SH-SY5Y cells derived from human bone marrow present a sub-clone of neuroblastoma line, with their transformation into neuron-like cells showing a range of highly instructive neurobiological characteristics. We applied two-step retinoic acid (RA) and brain-derived neurotrophic factor (BDNF) protocol to monitor the conversion of undifferentiated SH-SY5Y into neuron-like cells with distinctly polarized axon-dendritic morphology and formation of bona fide synaptic connections. We show that BDNF is a key driver and regulator of the expression of axonal marker tau and dendritic microtubule-associated protein-2 (MAP2), with their sorting to distinct cellular compartments. Using selective kinase inhibitors downregulating BDNF-TrkB signaling, we demonstrate that constitutive activation of TrkB receptor is essential for the maintenance of established polarization morphology. Importantly, the proximity ligation assay applied in our preparation demonstrates that differentiating neuron-like cells develop elaborate synaptic connections enriched with hallmark pre- and postsynaptic proteins. Described herein findings highlight several fundamental processes related to neuronal polarization and synaptogenesis in human-derived cells, which are of major relevance to neurobiology and translational neuroscience.
- MeSH
- biologické markery MeSH
- buněčná diferenciace genetika MeSH
- lidé MeSH
- mozkový neurotrofický faktor genetika metabolismus MeSH
- nádorové buněčné linie MeSH
- neuroblastom genetika metabolismus patologie MeSH
- neurogeneze genetika MeSH
- neurony cytologie metabolismus MeSH
- reaktivní formy kyslíku MeSH
- signální transdukce MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The nucleus-encoded 17β-hydroxysteroid dehydrogenase type 10 (17β-HSD10) regulates cyclophilin D (cypD) in the mitochondrial matrix. CypD regulates opening of mitochondrial permeability transition pores. Both mechanisms may be affected by amyloid β peptides accumulated in mitochondria in Alzheimer's disease (AD). In order to clarify changes occurring in brain mitochondria, we evaluated interactions of both mitochondrial proteins in vitro (by surface plasmon resonance biosensor) and detected levels of various complexes of 17β-HSD10 formed in vivo (by sandwich ELISA) in brain mitochondria isolated from the transgenic animal model of AD (homozygous McGill-R-Thy1-APP rats) and in cerebrospinal fluid samples of AD patients. By surface plasmon resonance biosensor, we observed the interaction of 17β-HSD10 and cypD in a direct real-time manner and determined, for the first time, the kinetic parameters of the interaction (ka 2.0 × 105 M1s-1, kd 5.8 × 104 s-1, and KD 3.5 × 10-10 M). In McGill-R-Thy1-APP rats compared to controls, levels of 17β-HSD10-cypD complexes were decreased and those of total amyloid β increased. Moreover, the levels of 17β-HSD10-cypD complexes were decreased in cerebrospinal fluid of individuals with AD (in mild cognitive impairment as well as dementia stages) or with Frontotemporal lobar degeneration (FTLD) compared to cognitively normal controls (the sensitivity of the complexes to AD dementia was 92.9%, that to FTLD 73.8%, the specificity to AD dementia equaled 91.7% in a comparison with the controls but only 26.2% with FTLD). Our results demonstrate the weakened ability of 17β-HSD10 to regulate cypD in the mitochondrial matrix probably via direct effects of amyloid β. Levels of 17β-HSD10-cypD complexes in cerebrospinal fluid seem to be the very sensitive indicator of mitochondrial dysfunction observed in neurodegeneration but unfortunately not specific to AD pathology. We do not recommend it as the new biomarker of AD.
- MeSH
- 17-hydroxysteroidní dehydrogenasy mozkomíšní mok metabolismus MeSH
- Alzheimerova nemoc metabolismus MeSH
- amyloidový prekurzorový protein beta genetika MeSH
- kinetika MeSH
- lidé MeSH
- mitochondrie metabolismus MeSH
- mozek metabolismus MeSH
- peptidylprolylisomerasa F metabolismus MeSH
- potkani transgenní MeSH
- potkani Wistar MeSH
- povrchová plasmonová rezonance MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
In Alzheimer's disease (AD), tau pathology manifested by the accumulation of intraneuronal tangles and soluble toxic oligomers emerges as a promising therapeutic target. Multiple anti-tau antibodies inhibiting the formation and propagation of cytotoxic tau or promoting its clearance and degradation have been tested in clinical trials, albeit with the inconclusive outcome. Antibodies against tau protein have been documented both in the brain circulatory system and at the periphery, but their origin and role under normal conditions and in AD remain unclear. While it is tempting to assign them a protective role in regulating tau level and removal of toxic variants, the supportive evidence remains sporadic, requiring systematic analysis and critical evaluation. Herein, we review recent data showing the occurrence of tau-reactive antibodies in the brain and peripheral circulation and discuss their origin and significance in tau clearance. Based on the emerging evidence, we cautiously propose that impairments of tau clearance at the periphery by humoral immunity might aggravate the tau pathology in the central nervous system, with implication for the neurodegenerative process of AD.
- MeSH
- Alzheimerova nemoc etiologie metabolismus patologie terapie MeSH
- autoantigeny imunologie MeSH
- autoprotilátky krev imunologie MeSH
- imunoterapie MeSH
- intravenózní imunoglobuliny terapeutické užití MeSH
- lidé MeSH
- náchylnost k nemoci * MeSH
- proteiny tau imunologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
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 Alzheimer's 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.
- MeSH
- Alzheimerova nemoc krev imunologie patologie terapie MeSH
- fosforylace MeSH
- imunoglobulin G krev klasifikace MeSH
- intravenózní imunoglobuliny krev imunologie metabolismus MeSH
- lidé středního věku MeSH
- lidé MeSH
- mozek metabolismus MeSH
- proteiny tau imunologie MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Increased 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. METHODS: MAO-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. RESULTS: Immunohistochemistry 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. CONCLUSIONS: This 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.
- MeSH
- Alzheimerova nemoc metabolismus patologie MeSH
- amyloidní beta-protein metabolismus MeSH
- axony metabolismus MeSH
- dendrity metabolismus MeSH
- krysa rodu rattus MeSH
- lidé středního věku MeSH
- lidé MeSH
- malá interferující RNA genetika metabolismus MeSH
- molekulární modely MeSH
- monoaminoxidasa genetika metabolismus MeSH
- mozek metabolismus patologie MeSH
- myši MeSH
- neurony metabolismus ultrastruktura MeSH
- presenilin-1 genetika MeSH
- receptory N-methyl-D-aspartátu metabolismus MeSH
- regulace genové exprese genetika MeSH
- sekretasy metabolismus MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- synapse metabolismus MeSH
- transfekce MeSH
- transformované buněčné linie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- senioři nad 80 let MeSH
- senioři MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Natural antibodies are now widely studied for their therapeutical potential. Therefore, their isolation and subsequent characterization is desired. Here, we describe an isolation method for natural anti-tau antibodies from human plasma by utilization of affinity chromatography with epoxy-activated copolymer resin. The evalution of isolation efficiency and avidity of isolated antibodies is decribed by modified indirect ELISA assay.
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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 Alzheimer's 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.
- MeSH
- adhezivita MeSH
- adsorpce MeSH
- Alzheimerova nemoc diagnóza MeSH
- aminokyselinové motivy MeSH
- biologické markery chemie MeSH
- chymotrypsin chemie MeSH
- epitopy chemie MeSH
- hmotnostní spektrometrie metody MeSH
- lidé MeSH
- magnetismus MeSH
- monoklonální protilátky chemie MeSH
- proteiny tau chemie MeSH
- proteolýza MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- thiazoly chemie MeSH
- trypsin chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
