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

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

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

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 Alzheimer's 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.

• MeSH
• epitopy chemie   MeSH
• imunoglobulin G imunologie   izolace a purifikace   MeSH
• imunosorpční techniky MeSH
• intravenózní imunoglobuliny chemie   imunologie   MeSH
• lidé MeSH
• molekulová hmotnost MeSH
• peptidy imunologie   MeSH
• proteiny tau imunologie   MeSH
• terciární struktura proteinů MeSH
• vazba proteinů imunologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH