The EMBO Meeting 2013

University of Zurich

Mechanisms of prion-induced toxicity

Prions cause neurodegeneration in vivo, yet prion-infected cultured cells are asymptomatic. This has hampered mechanistic studies of prion-induced neurodegeneration. We have found that prion-infected cultured organotypic cerebellar slices (COCS) experienced progressive spongiform neurodegeneration closely reproducing prion disease, with three different prion strains giving rise to three distinct patterns of prion protein deposition. Neurodegeneration did not occur when the Prnp gene was genetically removed from neurons, and a comprehensive pharmacological screen indicated that was abrogated by compounds known to antagonize prion replication. Prion infection of COCS led to enhanced fodrin cleavage, suggesting the involvement of calpains or caspases in pathogenesis. Accordingly, neurotoxicity and fodrin cleavage were prevented by calpain inhibitors but not by caspase inhibitors, whereas prion replication proceeded unimpeded. Hence calpain inhibition can uncouple prion replication from its neurotoxic sequelae. These data validate COCS as a powerful model system that faithfully reproduces most morphological hallmarks of prion infections. The exquisite accessibility of COCS to pharmacological manipulations was instrumental in recognizing the role of calpains in neurotoxicity, and significantly extends the collection of tools necessary for rigorously dissecting prion pathogenesis. The cellular prion protein PrPC consists of a globular domain (GD) hinged to a long N-proximal flexible tail (FT). We found rapid neurodegeneration in mice and in COCS exposed to holoantibodies, monovalent F(ab)1 fragments, or single-chain miniantibodies targeting the α1 and α3 helices of the GD. Degeneration was prevented by interstitial deletions within the FT and by treatment with various FT ligands, indicating that GD ligand toxicity was executed by the FT. Antibodies to the FT also prolonged the life of mice expressing a toxic PrPC mutant (PrPΔ94 134). These data uncover an essential role for the FT in two models of prion-related toxicity, and indicate that the FT triggers shared downstream effectors of neurodegeneration.

Biography

Adriano Aguzzi, MD, PhD, DVM hc, is Professor and Director of the Institute of Neuropathology at the University Hospital in Zurich. In the past 20 years he has focused entirely on prions, exploring how they reach and damage the brain after entering the body, and developing diagnostic and therapeutic approaches. As the Founding Director of the Swiss National Reference Center for Prion Diseases, Adriano Aguzzi has advised the British, Italian, and Swiss governments during the Mad Cow Disease crisis. He is a board member of the journal "Science", and sits on the scientific advisory board of several philanthropic foundations and biomedical companies. Adriano Aguzzi has received three honorary doctorates and an Advanced Grant of the European Research Council. He is an EMBO Gold Medal winner and a Fellow of the American Association for the Advancement of Science.

UCLA-DOE, Los Angeles

Two structure-based hypotheses about amyloid fibers and oligomers

From ~115 x-ray derived atomic structures of segments of prions and other amyloid-forming proteins, two hypothesizes have emerged:

The steric zipper hypothesis: the short adhesive segments of amyloid fibers are pairs of interdigitated β-sheets, called steric zippers. These can be inhibited, preventing fiber formation.

The cylindrin hypothesis: the oligomeric, etiologic agents of neurodegenerative amyloid diseases are anti-parallel, out-of-register β-sheets, possibly rolled into cylinders—cylindrins.

Toxic oligomers may be formed from anti-parallel out-of-register beta sheets, in which each pair of strands in a sheet is skewed relative to the pair below. This is the pattern we have observed in three structures which are cytotoxic, as judged by the MTT assay. In-register steric zippers stack each pair of strands exactly above the pair below along the fibril axis.

We have devised a structure-based procedure for design of non-natural peptides that inhibit fiber growth. We have also devised a combined structure and computational procedure for identification of small molecules that inhibit amyloid toxicity.

Our tentative conclusion is that there are separate amyloid aggregation pathways for in-register structures and out-of-register structures. The out-of-register structures are of higher energy because they have unsatisfied H-bonds, and hence are rarer. The higher-energy, out-of-register structures tend to form lower-energy, n-register structures over time, probably passing through the monomeric state. A high energy barrier separates out-of-register from in-register structures, because the transition must involve breaking and reforming many H-bonds. The design of small molecules that bind to in-register sheets tends to stabilize such sheets and to diminish the fraction of out-of-register structures, thereby lowering toxicity.

Biography

As a Harvard undergraduate, David Eisenberg had the good fortune to be assigned to study with John T. Edsall, one of the pioneers of protein chemistry, who oriented David to his life’s work. As a Rhodes Scholar at Oxford, Eisenberg earned a D.Phil. in theoretical chemistry for study with Charles Coulson on hydrogen bonding in ice. Returning to the States, Eisenberg worked as a postdoctoral fellow with Walter Kauzmann, the discoverer of the hydrophobic interaction. Together they wrote a monograph, The Structure and Properties of Water, still in print after 44 years. In further postdoctoral study at Caltech, Eisenberg learned X-ray crystallography. Since 1969, Eisenberg has been on the faculty of UCLA, now as the Paul D. Boyer Professor of Biochemistry and Molecular Biology, Investigator of the Howard Hughes Medical Institute, and Co-Director of the Center for Global Mentoring. Eisenberg now concentrates on proteins in the amyloid state. He has coauthored a text Physical Chemistry for Students of the Life Sciences, as well as about 300 research papers. Eisenberg belongs to several scholarly societies, including the U.S. National Academy of Sciences, the American Academy of Arts and Sciences, the Institute of Medicine, and the Philosophical Society.

ETH, Zurich

The Relationship between the 3D structures and Properties of Amyloids

Amyloids are highly ordered cross-β-sheet containing protein aggregates associated with several dozens diseases including Alzheimer’s, Creutzfeldt-Jakob and Parkinson’s disease, but are also associated with functional states such as hormone storage in secretory granules and skin pigmentation in mammals. In contrast to soluble protein folds, the cross-β-sheet entity is an inter-molecular motif repetitive in nature almost indefinitely. The intermolecular repetitiveness is key for the multiple properties of amyloids: On the one hand the repeating motifs can translate a rather non-specific interaction into a specific one through cooperativity. On the other hand the cross-β-sheet entity can grow by recruitment of the corresponding amlyoid peptide/proteins. Because of these two properties activities of amyloids are manifold including peptide storage, template assistant, loss of function, gain of function, generation of toxicity, membrane binding, infectivity etc. In this presentation, we will discuss the structure-function relationship of the HET-s prion of the filamentous fungus Podospera anserina, the role of the amyloid entity in the storage of hormones in secretory granules, and a possible mechanism of toxicity in α-synuclein associated Parkinson’s disease.

Biography

Roland Riek has been Full Professor of Physical Chemistry at ETH Zurich since May 2007. His group conducts research in the field of Biological NMR.

Roland Riek was born in Bern in 1969. He studied Physics at ETH Zurich. After earning his diploma, he remained at ETH to do his PhD with Prof. Kurt Wüthrich at the Institute for Molecular Biology and Biophysics. He finished his PhD thesis about the NMR structure of the Mouse Prion Protein in 1998. After a few years as postdoc at the Institute for Molecular Biology and Biophysics at ETH, he joined the structural biology laboratory of the Salk Institute for Biological Studies in La Jolla, California, first, in 2001, as assistant professor, then in 2006 as associate professor, and director of the NMR facility.

Research interests include the study of 3D structures of membrane proteins and misfolded protein aggregates associated with illnesses as well as the study of the dynamics of proteins and their interactions with other proteins.