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The Time- and Spatially Resolved Aggregation of a-Synuclein and its Relationship to Cell-Cell Transmissibility

English title The Time- and Spatially Resolved Aggregation of a-Synuclein and its Relationship to Cell-Cell Transmissibility
Applicant Riek Roland
Number 154461
Funding scheme Sinergia
Research institution Laboratorium für Physikalische Chemie D-CHAB ETH Zürich
Institution of higher education ETH Zurich - ETHZ
Main discipline Biophysics
Start/End 01.10.2014 - 31.03.2018
Approved amount 2'160'552.00
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All Disciplines (3)

Discipline
Biophysics
Physical Chemistry
Cellular Biology, Cytology

Keywords (4)

Parkinsons`s disease; Structural Biology; Transmissibility; Amyloid

Lay Summary (German)

Lead
Protein Aggregation ist ein Prozess, bei dem identische Proteine zu makroskopischen für den Körper toxischen Agglomeraten verklumpen. Dieser Aggregationsprozess wird mit ein paar Dutzend Krankheiten verknüpft, unter anderem Alzheimer und Parkinson Erkrankung.
Lay summary
Bei der Parkinson Erkrankung ist es das Protein α-synuclein, welches selbst aggregiert, respektive verklumpt. Mit diesem interdisziplinären Forschungsgesuch wird der Verlauf der Aggregation von α-synuclein sowohl im Reagenzglas als auch in der Zelle einerseits biophysikalisch, andererseits auch zellbiologisch untersucht. Ein wichtiger Aspekt  wird dabei sein, wie das verklumpte α-synuclein von einer Zelle zur anderen übertragen wird und sich selber vervielfältigt. Die Studien werden unter zu Hilfenahme von Kern Spin Resonanz Spektroskopie (NMR), Elektronenmikroskopie (EM), Super-Resolution Fluoreszenz Mikroskopie und diverse zellbiologische Techniken studiert. Mit dieser einmaligen Kombination von verschiedenen Techniken werden detaillierte Einsichten über die Beziehung zwischen Protein Aggregation und Proteinstruktur mit der Toxizität und Übertragung zwischen Zellen erlangt.
Direct link to Lay Summary Last update: 19.08.2014

Responsible applicant and co-applicants

Employees

Publications

Publication
The Microglial Innate Immune Receptor TREM2 Is Required for Synapse Elimination and Normal Brain Connectivity
Filipello Fabia, Morini Raffaella, Corradini Irene, Zerbi Valerio, Canzi Alice, Michalski Bernadeta, Erreni Marco, Markicevic Marija, Starvaggi-Cucuzza Chiara, Otero Karel, Piccio Laura, Cignarella Francesca, Perrucci Fabio, Tamborini Matteo, Genua Marco, Rajendran Lawrence, Menna Elisabetta, Vetrano Stefania, Fahnestock Margaret, Paolicelli Rosa Chiara, Matteoli Michela (2018), The Microglial Innate Immune Receptor TREM2 Is Required for Synapse Elimination and Normal Brain Connectivity, in Immunity, 48(5), 979-991.e8.
Miniaturizing EM Sample Preparation: Opportunities, Challenges, and “Visual Proteomics”
Arnold Stefan A., Müller Shirley A., Schmidli Claudio, Syntychaki Anastasia, Rima Luca, Chami Mohamed, Stahlberg Henning, Goldie Kenneth N., Braun Thomas (2018), Miniaturizing EM Sample Preparation: Opportunities, Challenges, and “Visual Proteomics”, in PROTEOMICS, 18(5-6), 1700176-1700176.
Microglia-Mediated Synapse Loss in Alzheimer's Disease
Rajendran Lawrence, Paolicelli Rosa C. (2018), Microglia-Mediated Synapse Loss in Alzheimer's Disease, in The Journal of Neuroscience, 38(12), 2911-2919.
A Map of Protein-Metabolite Interactions Reveals Principles of Chemical Communication.
Piazza I, Kochanowski K, Cappelletti V, Fuhrer T, Noor E, Saurer U, Picotti. P (2018), A Map of Protein-Metabolite Interactions Reveals Principles of Chemical Communication., in Cell, ''-''.
Cryo-EM analysis of homodimeric full-length LRRK2 and LRRK1 protein complexes
Sejwal Kushal, Chami Mohamed, Rémigy Hervé, Vancraenenbroeck Renée, Sibran William, Sütterlin Rosmarie, Baumgartner Paul, McLeod Robert, Chartier-Harlin Marie-Christine, Baekelandt Veerle, Stahlberg Henning, Taymans Jean-Marc (2017), Cryo-EM analysis of homodimeric full-length LRRK2 and LRRK1 protein complexes, in Scientific Reports, 7(1), 8667-8667.
Membrane Association Landscape of Myelin Basic Protein Portrays Formation of the Myelin Major Dense Line
Raasakka Arne, Ruskamo Salla, Kowal Julia, Barker Robert, Baumann Anne, Martel Anne, Tuusa Jussi, Myllykoski Matti, Bürck Jochen, Ulrich Anne S., Stahlberg Henning, Kursula Petri (2017), Membrane Association Landscape of Myelin Basic Protein Portrays Formation of the Myelin Major Dense Line, in Scientific Reports, 7(1), 4974-4974.
Lipid Internal Dynamics Probed in Nanodiscs
Martinez Denis, Decossas Marion, Kowal Julia, Frey Lukas, Stahlberg Henning, Dufourc Erick J., Riek Roland, Habenstein Birgit, Bibow Stefan, Loquet Antoine (2017), Lipid Internal Dynamics Probed in Nanodiscs, in ChemPhysChem, 18(19), 2651-2657.
TDP-43 Depletion in Microglia Promotes Amyloid Clearance but Also Induces Synapse Loss
Paolicelli Rosa C., Jawaid Ali, Henstridge Christopher M., Valeri Andrea, Merlini Mario, Robinson John L., Lee Edward B., Rose Jamie, Appel Stanley, Lee Virginia M.-Y., Trojanowski John Q., Spires-Jones Tara, Schulz Paul E., Rajendran Lawrence (2017), TDP-43 Depletion in Microglia Promotes Amyloid Clearance but Also Induces Synapse Loss, in Neuron, 95(2), 297-308.e6.
Blotting-free and lossless cryo-electron microscopy grid preparation from nanoliter-sized protein samples and single-cell extracts
Arnold Stefan A., Albiez Stefan, Bieri Andrej, Syntychaki Anastasia, Adaixo Ricardo, McLeod Robert A., Goldie Kenneth N., Stahlberg Henning, Braun Thomas (2017), Blotting-free and lossless cryo-electron microscopy grid preparation from nanoliter-sized protein samples and single-cell extracts, in Journal of Structural Biology, 197(3), 220-226.
Solution structure of discoidal high-density lipoprotein particles with a shortened apolipoprotein A-I
Bibow Stefan, Polyhach Yevhen, Eichmann Cédric, Chi Celestine N, Kowal Julia, Albiez Stefan, McLeod Robert A, Stahlberg Henning, Jeschke Gunnar, Güntert Peter, Riek Roland (2017), Solution structure of discoidal high-density lipoprotein particles with a shortened apolipoprotein A-I, in Nature Structural & Molecular Biology, 24(2), 187-193.
Amyloid Fibril Polymorphism: Almost Identical on the Atomic Level, Mesoscopically Very Different
Seuring Carolin, Verasdonck Joeri, Ringler Philippe, Cadalbert Riccardo, Stahlberg Henning, Böckmann Anja, Meier Beat H., Riek Roland (2017), Amyloid Fibril Polymorphism: Almost Identical on the Atomic Level, Mesoscopically Very Different, in The Journal of Physical Chemistry B, 121(8), 1783-1792.
Cell-wide analysis of protein thermal unfolding reveals determinants of thermostability.
Leuenberger P, Ganscha S, Kahraman A, Cappelletti V, Boersema PJ, von Mering C, Claassen M, Picotti P (2017), Cell-wide analysis of protein thermal unfolding reveals determinants of thermostability., in Science, ''-''.
FKBP12 contributes to α-synuclein toxicity by regulating the calcineurin-dependent phosphoproteome.
Caraveo G, Soste M, Cappelletti V, Fanning S, van Rossum DB, Whitesell L, Huang Y, Chung CY, Baru V, Zaichick S, Picotti P, Linquist S (2017), FKBP12 contributes to α-synuclein toxicity by regulating the calcineurin-dependent phosphoproteome., in Proc Natl Acad Sci U S A, ''-''.
Measuring protein structural changes on a proteome-wide scale using limited proteolysis-coupled mass spectrometry.
Schopper S, Kahraman A, Leuenberger P, Feng Y, Piazza I, Müller O, Boersema PJ, Picotti P (2017), Measuring protein structural changes on a proteome-wide scale using limited proteolysis-coupled mass spectrometry., in Nat.protoc., 2391-2410.
Evidence-Based Clinical Use of Nanoscale Extracellular Vesicles in Nanomedicine
Fais Stefano, O’Driscoll Lorraine, Borras Francesc E., Buzas Edit, Camussi Giovanni, Cappello Francesco, Carvalho Joana, Cordeiro da Silva Anabela, Del Portillo Hernando, El Andaloussi Samir, Ficko Trček Tanja, Furlan Roberto, Hendrix An, Gursel Ihsan, Kralj-Iglic Veronika, Kaeffer Bertrand, Kosanovic Maja, Lekka Marilena E., Lipps Georg, Logozzi Mariantonia, Marcilla Antonio, Sammar Marei, Llorente Alicia, Nazarenko Irina, et al. (2016), Evidence-Based Clinical Use of Nanoscale Extracellular Vesicles in Nanomedicine, in ACS Nano, 10(4), 3886-3899.
Preparation and Characterization of Stable α-Synuclein Lipoprotein Particles
Eichmann Cédric, Campioni Silvia, Kowal Julia, Maslennikov Innokentiy, Gerez Juan, Liu Xiaoxia, Verasdonck Joeri, Nespovitaya Nadezhda, Choe Senyon, Meier Beat H., Picotti Paola, Rizo Josep, Stahlberg Henning, Riek Roland (2016), Preparation and Characterization of Stable α-Synuclein Lipoprotein Particles, in Journal of Biological Chemistry, 291(16), 8516-8527.
Specific Inhibition of β-Secretase Processing of the Alzheimer Disease Amyloid Precursor Protein
Ben Halima Saoussen, Mishra Sabyashachi, Raja K. Muruga Poopathi, Willem Michael, Baici Antonio, Simons Kai, Brüstle Oliver, Koch Philipp, Haass Christian, Caflisch Amedeo, Rajendran Lawrence (2016), Specific Inhibition of β-Secretase Processing of the Alzheimer Disease Amyloid Precursor Protein, in Cell Reports, 14(9), 2127-2141.
The lipidome associated with the -secretase complex is required for its integrity and activity
Ayciriex S., Gerber H., Osuna G. M. G., Chami M., Stahlberg H., Shevchenko A., Fraering P. C. (2016), The lipidome associated with the -secretase complex is required for its integrity and activity, in Biochemical Journal, 473(3), 321-334.
Preparation and Characterization of Stable α-Synuclein Lipoprotein Particles
Eichmann Cédric, Campioni Silvia, Kowal Julia, Maslennikov Innokentiy, Gerez Juan, Liu Xiaoxia, Verasdonck Joeri, Nespovitaya Nadezhda, Choe Senyon, Meier Beat H., Picotti Paola, Rizo Josep, Stahlberg Henning, Riek Roland (2016), Preparation and Characterization of Stable α-Synuclein Lipoprotein Particles, in Journal of Biological Chemistry, 291(16), 8516-8527.
The activities of amyloids from a structural perspective
Riek Roland, Eisenberg David S. (2016), The activities of amyloids from a structural perspective, in Nature, 539(7628), 227-235.
The Three-Dimensional Structures of Amyloids
Riek Roland (2016), The Three-Dimensional Structures of Amyloids, in Cold Spring Harbor Perspectives in Biology, a023572-a023572.
Structure based aggregation studies reveal the presence of helix-rich intermediate during α-Synuclein aggregation
Ghosh Dhiman, Singh Pradeep K., Sahay Shruti, Jha Narendra Nath, Jacob Reeba S., Sen Shamik, Kumar Ashutosh, Riek Roland, Maji Samir K. (2015), Structure based aggregation studies reveal the presence of helix-rich intermediate during α-Synuclein aggregation, in Scientific Reports, 5, 9228-9228.
Emerging Roles of Extracellular Vesicles in the Nervous System
Rajendran L., Bali J., Barr M. M., Court F. A., Kramer-Albers E.-M., Picou F., Raposo G., van der Vos K. E., van Niel G., Wang J., Breakefield X. O. (2014), Emerging Roles of Extracellular Vesicles in the Nervous System, in Journal of Neuroscience, 34(46), 15482-15489.
Membrane Remodelling Activity of α-Synuclein
Riek Roland, Campioni Silvia, Membrane Remodelling Activity of α-Synuclein, in J Neurol Neuromed, 1, 23-27.

Collaboration

Group / person Country
Types of collaboration
Omar Mukhtar El-Agnaf, UAE United Arab Emirates (Asia)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Dr. Kostas Vekrellis Greece (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. M. Tolnay, Pathology, Medical Faculty, Uni Basel Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results

Associated projects

Number Title Start Funding scheme
164074 Cryo-Electron Microscopy in the ZMB of the University of Basel 01.06.2016 R'EQUIP
148329 In cell NMR study of a-Syn and its familial disease-associated mutants. 01.05.2013 International short research visits
144444 Structural Studies of Aggregates and Membrane Proteins 01.01.2013 Project funding (Div. I-III)
177195 Molecular and Cellular Modulation in Parkinson's Disease 01.01.2018 Sinergia

Abstract

The self-association of identical proteins into macroscopic entities with toxic and/or functional activities is an omnipresent process. Some protein aggregates exhibit a cross-ß-sheet structure and are associated with several human diseases including Alzheimer’s and Parkinson’s disease (PD), but may also have a non-pathological function. This interdisciplinary proposal aims to obtain a detailed mechanistic understanding of the structural and cellular basis of the cell-to-cell transmissibility of a-synuclein (a-syn) aggregates, which are associated with PD. In particular it focuses on in vitro and in-cell studies of the spatially and time-resolved formation of seeds, the replication of amyloids, the role of the subcellular milieu in these processes, and the elucidation of environment-dependent amyloid polymorphisms and their relationship to transmissibility and toxicity. We propose to study the kinetics and structures of protein aggregation in vitro under a plethora of physiologically relevant conditions covering intracellular compartments (RR) and beyond, and to correlate these finding with the in-cell aggregation monitored by electron microscopy (EM) (HS) and super resolution fluorescence microscopy (SR) (JR), accompanied by cell biology studies on the transmissibility and the intracellular route of the infectious material (LR). Using this rather unique combination of techniques applied to both in vitro and in-cell studies, will give insight into (i) the relationships between the aggregation kinetics of a-syn, its localization to specific intracellular compartments and its cell-to-cell transmissibility and (ii) the relationship between cell toxicity and transmissibility.Aim 1: To achieve a quantitative phase-space analysis of a-syn aggregation in vitro and determine the role of additives in the aggregation process. In PBS buffer without an air-water interface, a-syn does not aggregate, while it readily produces polymorphic aggregates in the presence of negatively charged membranes. We will study the phase-space of a-syn aggregation under air-free conditions in diverse buffers mimicking the conditions of various subcellular compartments (RR), and in the presence and absence of additives, e.g., membranes and glycosaminoglycans (LR). This will be achieved using a Thioflavin T-based 96 well assays accompanied by quantitative, microfluidics-based EM (HS) and SR (JR). We will determine a-syn aggregate structures by biophysical techniques, incl. NMR, to determine the environment-defined structural landscape (RR).Aim 2: To determine the intracellular aggregation kinetics of a-syn. Microfluidics-based quantitative EM methods will be used to quantitatively analyze the aggregation kinetics of a-syn in cells (HS) and accompanied by in-cell SR (JR). The knowledge will be completed by in-cell NMR studies of the aggregation kinetics (RR), and by measuring seed-based aggregation time courses in cells and in vitro. Further, cell biology methods that interfere with the aggregation process in cells by inhibiting certain pathways or trafficking routes, will be used to explore the intra-cellular route of the various a-syn entities, and elucidate the place of (i) nucleus formation and (ii) aggregate replication (LR).Aim 3: To understand the uptake and release of a-syn aggregates: The cellular mechanisms underlying transmissibility of a-syn aggregates will be investigated using cell biological and systems biology tools (LR). In particular, the release and uptake of a-syn amyloids (from recombinant sources and cell derived) will be explored by the combination of EM (HS), SR (JR) and cell biological techniques interfering with subcellular compartment trafficking (LR).Aim 4: To get mechanistic insights correlating a-syn transmissibility and toxicity. The transmissibility of a-syn aggregates will be studied by cell biology methods (LR) and EM (HS). The toxicity of recombinant a-syn material as well as cell-derived a-syn will be evaluated using both cell-based assays (LR), and an EM-based analysis of the mesoscopic structure of the cell (LR, HS). The apparent connection between a-syn cell-to-cell transmission and toxicity will be studied using the highly toxic artificial variant E57K and the non-toxic but highly aggregation-prone a-syn(30-110) variant. The a-syn toxicity and transmissibility will be correlated to the in vitro kinetic studies (RR, aim 1) by using in vitro-generated a-syn material to help establish the structure-toxicity and a structure-infectivity relationship.
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