المؤلفون: Martin Kiechle, Bjoern von Einem, Lennart Höfs, Patrizia Voehringer, Veselin Grozdanov, Daniel Markx, Rosanna Parlato, Diana Wiesner, Benjamin Mayer, Olena Sakk, Bernd Baumann, Soeren Lukassen, Birgit Liss, Arif B. Ekici, Albert C. Ludolph, Paul Walther, Boris Ferger, Pamela J. McLean, Björn H. Falkenburger, Jochen H. Weishaupt, Karin M. Danzer

المصدر: Cell Reports, Vol 29, Iss 9, Pp 2862-2874.e9 (2019)

مصطلحات موضوعية: Biology (General), QH301-705.5

الوصف: Summary: Intracellular accumulation of α-synuclein (α-syn) and formation of Lewy bodies are neuropathological characteristics of Parkinson’s disease (PD) and related α-synucleinopathies. Oligomerization and spreading of α-syn from neuron to neuron have been suggested as key events contributing to the progression of PD. To directly visualize and characterize α-syn oligomerization and spreading in vivo, we generated two independent conditional transgenic mouse models based on α-syn protein complementation assays using neuron-specifically expressed split Gaussia luciferase or split Venus yellow fluorescent protein (YFP). These transgenic mice allow direct assessment of the quantity and subcellular distribution of α-syn oligomers in vivo. Using these mouse models, we demonstrate an age-dependent accumulation of a specific subtype of α-syn oligomers. We provide in vivo evidence that, although α-syn is found throughout neurons, α-syn oligomerization takes place at the presynapse. Furthermore, our mouse models provide strong evidence for a transsynaptic cell-to-cell transfer of de novo generated α-syn oligomers in vivo. : Kiechle et al. present two transgenic mouse models that allow direct quantitative and spatial detection of α-synuclein (α-syn) oligomers in vivo. They demonstrate α-syn aggregation at the synapse, age-dependent accumulation of 8–16-mer α-syn oligomer species, and trans-cellular oligomer spreading from forebrain to hindbrain neurons. Keywords: Parkinson's disease, transgenic mouse model, alpha-synuclein, oligomerization, synaptic oligomers, aging, neurodegeneration, progressive phenotype, spreading, protein-fragment complementation

وصف الملف: electronic resource

العلاقة: http://www.sciencedirect.com/science/article/pii/S2211124719314111Test; https://doaj.org/toc/2211-1247Test

الوصول الحر: https://doaj.org/article/351c4be491104a72a841cfbabe63feb7Test

المؤلفون: Barbara Moepps, Mariana Lang, Sandra Beier, Michael Abadier, Daniel Markx, Julia Schuhholz

المصدر: Cellular Signalling. 53:170-183

مصطلحات موضوعية: 0301 basic medicine, Chemokine, Cell signaling, RHOA, biology, Phospholipase C, Chemistry, Cell Biology, Cell biology, 03 medical and health sciences, Chemokine receptor, 030104 developmental biology, 0302 clinical medicine, Gq alpha subunit, 030220 oncology & carcinogenesis, biology.protein, CC chemokine receptors, Receptor

الوصف: In man, two CC chemokine receptor isoforms, CCR2a and CCR2b, are present that belong to the rhodopsin-like G protein-coupled receptor family, and couple to Gi and Gq family members. The CCR2 receptors are known to regulate canonical functions of chemokines such as directed migration of leukocytes, and to potentially control non-canonical functions such as differentiation, proliferation, and gene transcription of immune and non-immune cells. We recently reported on the activation of phospholipase C isoenzymes and RhoA GTPases by coupling of the two CCR2 receptors to members of the Gq family, in particular Gαq and Gα14. So far little is known about the structural requirements for the CCR2/Gq/14 interaction. Interestingly, the CCR2 receptor isoforms are identical up to arginine 313 (R313) that is part of the putative 8th helix in CCR2 receptors, and the 8th helix has been implicated in the interaction of rhodopsin-like G protein-coupled receptors with Gαq. In the present work we describe that the 8th helix of both CCR2a and CCR2b is critically involved in selectively activating Gαq/14-regulated signaling. Refined analysis using various CCR2a and CCR2b mutants and analyzing their cellular signaling, e.g. ligand-dependent (i) activation of phospholipase C isoenzymes, (ii) stimulation of serum response factor-mediated gene transcription, (iii) activation of mitogen-activated protein kinases, (iv) internalization, and (v) changes in intracellular calcium concentrations, identified arginine 313 within the amino terminal portion of helix 8 to play a role for the agonist-mediated conformational changes and the formation of a Gαq/14 binding surface. We show that R313 determines Gαq/14 protein-dependent but not Gi protein-dependent cellular signaling, and plays no role in Gq/Gi-independent receptor internalization, indicating a role of R313 in biased signaling of CCR2 receptors.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_________::79120b399b231907fc9cba07ad2dc569Test
https://doi.org/10.1016/j.cellsig.2018.10.007Test

المؤلفون: Marcus Fändrich, Daniel Markx, Christian Mawrin, Cornelia Loos, Stephanie Claus, Christian Haupt

المصدر: Biochemical and Biophysical Research Communications. 497:857-862

مصطلحات موضوعية: 0301 basic medicine, Amyloid, Protein Folding, Protein Conformation, Biophysics, Brain tissue, Fibril, Biochemistry, Protein Aggregates, 03 medical and health sciences, Alzheimer Disease, medicine, Humans, Prion protein, Molecular Biology, Brain Chemistry, Amyloid beta-Peptides, Chemistry, Neurodegeneration, Cell model, Aβ peptide, Brain, Cell Biology, medicine.disease, Peptide Fragments, 030104 developmental biology, Aβ amyloid, Protein folding

الوصف: Intracerebral injection of brain extracts from Alzheimer's disease (AD) patients into appropriate mouse models was previously found to drastically accelerate the deposition of Aβ amyloid in the recipient animals indicating a prion-like activity. In this study we show that this prion-like activity can be also identified by using a cell culture model of Aβ plaque formation. Analysis of biochemical fractions of AD brain extract indicate that the seeding-activity correlated with the presence of Aβ peptide and Aβ-derived aggregates. In vitro-formed fibrils were also active but their activity was low and depending on the fibril structure and conditions of fibril formation. Our data indicate a conformational basis of the observed seeding effect and suggest the utility of our cell model for further studies on the prion-like activity of AD extracts.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::0056fc946adfd44f12eb41dc6950c3ccTest
https://doi.org/10.1016/j.bbrc.2018.02.137Test

المؤلفون: Daniel, Markx, Julia, Schuhholz, Michael, Abadier, Sandra, Beier, Mariana, Lang, Barbara, Moepps

المصدر: Cellular signalling. 53

مصطلحات موضوعية: Protein Conformation, alpha-Helical, HEK293 Cells, Receptors, CCR2, COS Cells, Chlorocebus aethiops, Animals, GTP-Binding Protein alpha Subunits, Gq-G11, Humans, Protein Interaction Maps, Arginine, Signal Transduction

الوصف: In man, two CC chemokine receptor isoforms, CCR2a and CCR2b, are present that belong to the rhodopsin-like G protein-coupled receptor family, and couple to G

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=pmid________::0dfabb116128d536e7b49d5b46c3c5a7Test
https://pubmed.ncbi.nlm.nih.gov/30321592Test

المؤلفون: Daniel Markx, Paul Walther, Stefanie Sieste, Marcus Fändrich, Markus Lamla, Tanja Weil, Annika Röcker, Christoph Meier, Sascha Rode, Jan Münch, Manuel Hayn, Frank Kirchhoff

المصدر: Bioconjugate Chemistry

مصطلحات موضوعية: 0301 basic medicine, Biomedical Engineering, Nanofibers, Pharmaceutical Science, Bioengineering, Peptide, Fibril, Avian sarcoma virus, Virus, Viral vector, 03 medical and health sciences, Transduction (genetics), 0302 clinical medicine, Transduction, Genetic, Fluorescent Dyes, Pharmacology, chemistry.chemical_classification, Organic Chemistry, Gene Transfer Techniques, Molecular biology, In vitro, 030104 developmental biology, Membrane, Retroviridae, Spectrometry, Fluorescence, chemistry, Biophysics, Peptides, 030217 neurology & neurosurgery, Biotechnology

الوصف: Retroviral gene transfer is the method of choice for the stable introduction of genetic material into the cellular genome. However, efficient gene transfer is often limited by low transduction rates of the viral vectors. We have recently described a 12-mer peptide, termed EF-C, that forms amyloid-like peptide nanofibrils (PNF), strongly increasing viral transduction efficiencies. These nanofibrils are polycationic and bind negatively charged membranes of virions and cells, thereby overcoming charge repulsions and resulting in increased rates of virion attachment and gene transfer. EF-C PNF enhance vector transduction more efficiently than other soluble additives and offer prospects for clinical applications. However, while the transduction-enhancing activity of PNF has been well-characterized, the exact mechanism and the kinetics underlying infection enhancement as well as the cellular fate of the fibrils are hardly explored. This is partially due to the fact that current labeling techniques for PNF rely on amyloid probes that cause high background staining or lose signal intensities after cellular uptake. Here, we sought to generate EF-C PNF covalently coupled with fluorescent labels. To achieve such covalent bioconjugates, the free amino groups of the EF-C peptide were coupled to the ATTO 495 or 647N NHS ester dyes. When small amounts of the labeled peptides were mixed with a 100- to 10 000-fold excess of the native peptide, PNF formed that were morphologically indistinguishable from those derived from the unlabeled peptide. The fluorescence of the fibrils could be readily detected using fluorescence spectroscopy, microscopy, and flow cytometry. In addition, labeled and nonlabeled fibrils captured viral particles and increased retroviral transduction with similar efficacy. These covalently fluorescence-labeled PNF are valuable tools with which to elucidate the mechanism(s) underlying transduction attachment and the fate of the fibrils in cells, tissues, and animal models.

وصف الملف: application/pdf

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::0f1b3bb9803f996eb936cf7299464232Test
https://pubmed.ncbi.nlm.nih.gov/28300392Test

المؤلفون: Shen Han, Marius Kollmer, Daniel Markx, Stephanie Claus, Paul Walther, Marcus Fändrich

المصدر: Scientific Reports

مصطلحات موضوعية: Amyloid, Electron Microscope Tomography, Amyloid beta-Peptides, Cell Survival, Cell Membrane, Plaque, Amyloid, macromolecular substances, Lipids, Protein Aggregation, Pathological, Article, Protein Aggregates, Alzheimer Disease, Humans, Cells, Cultured

الوصف: The deposition of amyloid fibrils as plaques is a key feature of several neurodegenerative diseases including in particular Alzheimer's. This disease is characterized, if not provoked, by amyloid aggregates formed from Aβ peptide that deposit inside the brain or are toxic to neuronal cells. We here used scanning transmission electron microscopy (STEM) to determine the fibril network structure and interactions of Aβ fibrils within a cell culture model of Alzheimer's disease. STEM images taken from the formed Aβ amyloid deposits revealed three main types of fibril network structures, termed amorphous meshwork, fibril bundle and amyloid star. All three were infiltrated by different types of lipid inclusions from small-sized exosome-like structures (50-100 nm diameter) to large-sized extracellular vesicles (up to 300 nm). The fibrils also presented strong interactions with the surrounding cells such that fibril bundles extended into tubular invaginations of the plasma membrane. Amyloid formation in the cell model was previously found to have an intracellular origin and we show here that it functionally destroys the integrity of the intracellular membranes as it leads to lysosomal leakage. These data provide a mechanistic link to explain why intracellular fibril formation is toxic to the cell.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=pmid_dedup__::8faafa6a7f250db55505485bdceb1bacTest
http://europepmc.org/articles/PMC5327471Test

المؤلفون: Megan Garvey, Senthil Kumar, Melanie Wulff, Marcus Fändrich, Jochen Balbach, Isabel Morgado, Christian Mawrin, Daniel Markx, Uwe Knüpfer, Monika Baumann, Uwe Horn

المصدر: Amyloid : the international journal of experimental and clinical investigation : the official journal of the International Society of Amyloidosis. 23(2)

مصطلحات موضوعية: 0301 basic medicine, Pathology, medicine.medical_specialty, Amyloid, Protein Folding, Protein Conformation, Plaque, Amyloid, macromolecular substances, Buffers, Fibril, 01 natural sciences, Models, Biological, Cell Line, Phosphates, 03 medical and health sciences, Protein structure, Internal Medicine, medicine, Humans, Nuclear Magnetic Resonance, Biomolecular, Conserved Sequence, Amyloid beta-Peptides, 010405 organic chemistry, Chemistry, Neurodegeneration, P3 peptide, Deuterium Exchange Measurement, medicine.disease, In vitro, Peptide Fragments, 0104 chemical sciences, Solutions, 030104 developmental biology, Solid-state nuclear magnetic resonance, Cell culture, Biophysics, Protein folding

الوصف: Objectives: The detailed structure of brain-derived Aβ amyloid fibrils is unknown. To approach this issue, we investigate the molecular architecture of Aβ(1–40) fibrils grown in either human cerebrospinal fluid solution, in chemically simple phosphate buffer in vitro or extracted from a cell culture model of Aβ amyloid plaque formation.Methods: We have used hydrogen–deuterium exchange (HX) combined with nuclear magnetic resonance, transmission electron microscopy, seeding experiments both in vitro and in cell culture as well as several other spectroscopic measurements to compare the morphology and residue-specific conformation of these different Aβ fibrils.Results and conclusions: Our data reveal that, despite considerable variations in morphology, the spectroscopic properties and the pattern of slowly exchanging backbone amides are closely similar in the fibrils investigated. This finding implies that a fundamentally conserved molecular architecture of Aβ peptide fold is common to Aβ fibrils.

الوصول الحر: https://explore.openaire.eu/search/publication?articleId=doi_dedup___::7c3b20cf386845afe087fc1b1ec8a825Test
https://pubmed.ncbi.nlm.nih.gov/26972581Test