Dr. Collinge began with an overview of several topics including acquired prion disease in humans, the codon 129 polymorphism, the crystal structure of PrPC, and the concept of transmission barriers. He discussed several of MRC Prion Unit’s earlier findings regarding how genetic polymorphisms and transgenes can affect the transmission barrier [Collinge 1995, Wadsworth 2004, Lukic 2010]. Much of the first half of the talk was a documentary-style narrative on the history of kuru, featuring images of the Papua New Guinea landscape and videos of interviews with several Fore people. Dr. Collinge recalled that when he first started publishing on kuru, he began to receive angry letters from anthropologists who believed that kuru was an imperialist myth concocted to make the Fore look like savages. He likes to show the interviews in part to demonstrate that this isn’t the case. He showed some striking photographs of the field research station that MRC Prion Unit set up in the mountains of the Fore region, including a hand-powered centrifuge. He discussed the incredibly length incubation periods of kuru. Endocannibalism was phased out by ~1961, yet there was still a trickle of people falling ill with kuru in the late 1990s, and they even identified one new case last year. He discussed the way in which kuru selected for 129MV and 127VG heterozygotes [Mead 2009], and the evidence for worldwide balancing selection favoring heterozygosity for PRNP [Mead 2003]. He noted that the 129V allele has an allele frequency of ~55% in the Fore region, the highest allele frequency observed anywhere in the world.
Next he moved into some new results that are currently in press. He asked me not to share any of these results here, but he expects the paper will be published within a few weeks.
Dr. Strittmatter began with a review of his lab’s screen that identified PrPC as a high-affinity ligand for Aβ oligomers [Lauren 2009]. He also discussed his lab’s findings that Aβ binding to PrPC triggers a signaling pathway via Fyn and mGluR5 [Um 2012, Um 2013]. He discussed the effects of this binding event on long-term potentiation [Lauren 2009], which have been replicated by John Collinge’s group [Freir 2011]. They have used antibody binding to map the mGluR5 binding site on PrP, which appears to be within PrP residues 91-153 and binds preferentially to the activated state of mGluR5 [Haas 2014]. A small molecule allosteric antagonist of mGluR5, called MTEP, ameliorates behavioral deficits in APP/PS1 mice [Um 2013]. They have also tested a small molecule inhibitor of Fyn, called AZD0530 and originally developed for cancer, and found that it rescued synaptic and behavioral phenotypes [Kaufman 2015]. Based on these promising results, he initiated a multi-site clinical trial, NCT01864655, to test AZD0530 for treatment of Alzheimer disease.
He also presented a number of unpublished results.
Update 2015-06-02 They have also worked to characterize the species of Aβ that binds PrP, and it appears to be a high molecular weight species [Kostylev 2015].
David A. Harris
Dr. Harris began by reviewing his and other labs’ evidence that membrane-bound PrPC is required for transduction of a neurotoxic signal by PrPSc. To understand how PrP transduces this signal, it is necessary to consider the structure of PrP as divided into three sections: the unstructured N terminus, the “hinge region” (from the end of the octarepeats to the first beta sheet), and then the C-terminal globular domain [Biasini 2012]. He then listed the deletion mutants of PrP created by his lab and the Weissmann and Aguzzi labs, focusing on his ΔCR mutant [Li 2007], which gives the most dramatic phenotype. PrP ΔCR results in spontaneous inward transmembrane currents, which require residues 23-31 [Solomon 2010, Solomon 2011].
He quickly moved into a large volume of unpublished results that I cannot share here.
Dr. Krob began by reviewing the list of known prions in yeast, and introducing the [PSI+] model system (which I’ve also introduced here). In short, the incorporation of Sup35 protein into [PSI+] amyloid results in a loss of Sup35’s native translation termination function. In yeast with a stop codon in the ade gene grown on minimal adenine media, [psi-] colonies are red, while [PSI+] colonies are white. She reviewed Jonathan Weissman’s work characterizing strong vs. weak [PSI+] prions, which appear to have ~40 vs. ~70 amino acid cores. Strong [PSI+] prions are more thermolabile but also fragment more readily, creating a greater number of propagons, and thus are less readily cured [Tanaka 2006, Toyama 2007]. She then reviewed evidence from the Liebman lab regarding different [RNQ+] prions and how these have differential effects on [PSI+] seeding. (I’m not too familiar with that literature but she may have been referring to [Vitrenko 2007a, Vitrenko 2007b]). She then moved into her own lab’s work on how genetic and environmental factors affect the ability of [RNQ+] to seed [PSI+]. Some of this work appears to be published [Huang 2013, Westergard & True 2014, Stein & True 2014], but I am not sufficiently familiar with this literature to be certain of what is acceptable to blog here. She then discussed the establishment of DNAJB6 as a limb-girdle muscular dystrophy gene [Harms 2012]. DNAJB6 encodes an HSP40 chaperone, and Dr. True’s lab has used a yeast system to examine how the human mutations affect chaperone activity including recognition of two conformers of [RNQ+] and [PSI+] [Stein 2014].
Those who have been following Dr. Sigurdson’s work will know that she has been interested in the molecular basis of the prion strain barrier for chronic wasting disease, ever since her days in the Hoover lab and then the Aguzzi lab. She has amassed a variety of lines of evidence indicating that the β2-α2 loop in PrP governs susceptibility to CWD [Sigurdson 2009, Sigurdson 2010, Sigurdson 2011]. Since starting her own lab she has investigated the structural basis of this transmission barrier through additional mouse models and NMR studies [Kurt 2014a, Kurt 2014b, Kurt 2015]. After reviewing these published works, she went on to discuss unpublished results from new mouse models.
As an aside, Dr. Sigurdson also noted that she has started using an experimental system where cell lysate is a substrate for PMCA [Mays 2011]. And she talked about some interesting results on amyloid structure from the Eisenberg lab [Sawaya 2007, Goldschmidt 2010].
Dr. Andreoletti opened with a very brief overview of the importance of understanding the risk of prion transmission by blood transfusion, citing the U.K. appendix study [Gill 2013]. He acknowledged Fiona Houston’s important work, presented yesterday, and said that he is looking at similar questions using different animal models. He spent most of the talk presenting 8-year followup data on a study of transfusion risk in sheep and mouse models. I believe most of this was unpublished, though there was at least some overlap with results he published a few years ago [Andreoletti 2012]. He also spent a bit of time discussing his published results indicating that endpoint titration gives similar results in sheep or in mice overexpressing sheep PrP [Douet 2014], and on the effects of leukodepletion of blood products on prion titer [Lacroux 2012].
Dr. Chiesa reviewed his mouse models of PrP octapeptide repeat insertions [Chiesa 1998], D178N-129V [Dossena 2008], and D178N-129M [Bouybayoune 2015], and his prior work investigating the molecular basis of toxicity in these mouse models [Chiesa 2001, Quaglio 2011, Senatore 2012]. He presented unpublished results, which are summarized in his abstract O.09 here, following up on these lines of inquiry. He also noted that his previous finding that post-Golgi trafficking is impaired in cells expressing D178N-129M PrP [Bouybayoune 2015] mirror the findings from scrapie-infected cells [Uchiyama 2013].
I didn’t catch all of this talk but the part that I saw appeared to be mostly published work regarding the role of innate immunity and microglial activation in experimental prion disease [e.g. Cunningham 2009, Gomez-Nicola 2013]. He also discussed the results of a recent clinical trial of etanercept, a TNFα inhibitor, in Alzheimer disease [Butchart 2015].
Dr. Lasmezas spoke about the role of NAD+ starvation in prion-induced neurotoxicity. She presented on this topic at Prion2013 in Banff and the findings were recently published [Zhou 2015]. This line of inquiry started with efforts to develop a cellular model for prion neurotoxicity. By testing different fractions of recombinant PrP, Dr. Lasmezas identified a monomeric, alpha-helical species of PrP that causes death in cultured PK1 cells [Zhou 2012]. This species, dubbed TPrP, is toxic at only 20 nM. Nicotinamide, however, was found to rescue the toxicity. Nicotinamide is considered a vitamin, and is the precuror for NAD+. Adding FK866, an inhibitor of the phosphoribosyltransferase that makes NAD+ from nicotinamide, reverses the rescue of toxicity by nicotinamide. They then found that intracellular NAD+ and ATP levels decline after cells are exposed to TPrP. TPrP appears to cause excessive ADP-ribosylation independent of PARP1. Intranasal NAD+ administration to prion-infected mice was observed to improve behavioral phenotypes but had no effect on survival [Zhou 2015].
Dr. Allison used a zinc finger nuclease to engineer a targeted disruption of the PrP2 gene in zebrafish [Fleisch 2013]. They looked pretty hard for a phenotype in the homozygous PrP2 knockout fish but they did not see anything morphologically or behaviorally wrong with them at baseline. When the fish were exposed to a convulsant, however, they had increased susceptibility to seizures, and they observed electrophysiological differences in NMDA signaling [Fleisch 2013]. He noted that it has been reported that PrP regulates NMDA in mice, but that the effect requires copper [You 2012], whereas it appears that PrP2 in zebrafish does not bind copper - in place of the mammalian octapeptide repeat, it has a hexapeptide repeat (consensus PAQGGY) which lacks copper-binding histidines.
He next discussed his experiments knocking down zebrafish appb (an ortholog of APP), prp1, and prp2 with morpholinos [Kaiser 2012], and lateral line developmental phenotypes in PrP2 knockouts and knockdowns [Huc-Brandt 2014]. He also presented a few unpublished results from these systems.
He concluded by arguing that PrP has an ancient role in regulating neuronal excitability.
Q. Alex McDonald: Have you thought about using CRISPR-Cas to knock in a PrP mutant lacking the repeat domain or the polybasic tail?
A. It’s a great idea. CRISPR-Cas reagents for zebrafish have become available just over the past 9-12 months and we have started doing some experiments with them.
Q. Joel Watts: Is APP in the zebrafish processed the same way as it is in mammals?
A. No one has published any data on that, but the structure of zebrafish APP certainly would lead you to expect it to be cleaved in the same positions.
Dr. Altmeppen presented some of his work in Markus Glatzel’s lab that was recently published [Altmeppen 2015]. He introduced the concepts of PrP alpha cleavage and shedding, and said he would not discuss beta cleavage. He noted that these proteolytic events are conserved and probably constitutive, and may represent an important aspect of PrP’s native function. He examined a published Nestin-Cre neuronal ADAM10 conditional knockout (cKO) mouse [Jorissen 2010] and found that these mice lack PrP shedding, accumulate PrPC in the early secretory pathway, but have unimpaired alpha cleavage [Altmeppen 2011]. These data establish ADAM10 as the major sheddase for PrPC. The Jorissen ADAM10 conditional knockout mouse exhibits perinatal lethality, which made it impossible to assess the effects of ADAM10 deletion on prion disease progression. Therefore, in the new study, Dr. Altmeppen generated a new CamKIIα-Cre conditional knockout (cKO) mouse, which lacks ADAM10 only in forebrain neurons. These mice were i.c. inoculated with RML prions into the striatum. The homozygous and, to a lesser extent, heterozygous cKO mice had truncated incubation times compared to wild-type mice [Altmeppen 2015]. They also had increased levels of PrPC, perhaps due to the retention / lack of shedding characterized earlier [Altmeppen 2011]. However, Dr. Altmeppen believes that the shortened incubation time is probably not due solely to increased PrPC levels, because PrPSc levels were increased more dramatically than total PrP. He suspects that shed PrP may have a dual role, being capable both of spreading pathology but also of blocking PrPSc formation. He is now interested in in ADAM10 activation as a potential therapeutic target for treating prion diseases.
I missed almost all of this talk but according to the oral abstract it appears to have all been unpublished work.
This talk was all unpublished data; Dr. Manson said it would be publicly available soon.
Dr. Chiti’s talk sought to answer the question of what structural elements in amyloid conversion are involved in toxicity. Like in his Prion2014 talk, he used the HypF-N protein (the N-terminal domain of E. coli HypF protein) as a model system. He briefly reviewed his work in this area going back to 2001 [e.g. Chiti 2001, Campioni 2008] and state that HypF-N forms amyloid fibrils that are tinctorally and biochemically indistiguishable from the amyloid fibrils associated with disease, and are cytotoxic. He defined two types of HypF-N oligomers, of which only type A presents exposed hydrophobic surfaces and is cytotoxic [Campioni 2010, Zampagni 2011]. In primary cell cultures, slice cultures, and in rats, these oligomers exhibit many of the same properties as Aβ oligomers [Tatini 2013]. He discussed how chaperones suppress the toxicity of these oligomers. The chaperones appear to work to group the oligomers into large aggregates [Mannini & Cascella 2012]. Finally, he presented results from his most recent study, in which they mutated charged residues to hydrophobic residues and found that across the different mutants they generated, cytotoxicity was a function of hydrophobic surface area and size [Mannini 2014].
Q. Glenn Telling: How exactly do these things interact with cells in order to be toxic?
Q. Neil Cashman: Are you sure there is no difference in size between type A and B oligomers? You would expect tighter packing in type B, leading to a smaller diameter.
A. We’ve looked carefully using techniques that allow us to measure shape and height, but it would be useful to look with electron microscopy as well.
Karen Hsiao Ashe
Dr. Ashe opened with a quote:
This structural transition from α-helixes to β-sheet in PrP is the fundamental event underlying prion diseases.
She recalled her early work on GSS [Hsiao 1989, Hsiao 1990, Hsiao 1994], and noted that while GSS and Alzheimer disease both involve plaques made of transmissible proteins, in GSS fatal disease is transmitted, while in Alzheimer disease only the plaques are transmitted. Moreover, while “amyloid” is a distinctive feature of prion disease, the properties we refer to by this term are not actually necessary for infectivity [Wille 2000], raising the question of what the infectious species really is.
Dr. Ashe noted that 412 clinical trials have been registered for Alzheimer disease between 2002 and 2012, with a 99.6% failure rate.Is the failure rate so high for lack of fundamental scientific understanding of the disease process, or for lack of clinical application of the science we do understand? She urged us to be patient, noting that immune checkpoints were discovered 20 years ago, but our understanding of them has only begun to aid cancer therapeutics within the past few years [Topalian 2015].
Dr. Ashe refers to the unknown, non-amyloid species of protein responsible for transmissibility in various diseases as “protein*”. She drew a distinction between three protein species seen in neurodegenerative disease: amyloid, the transmissible protein* species, and the species responsible for neuronal impairment or death. She recalled her studies of a mouse model with tau under a Tet-off promoter [SantaCruz 2005]. After the mice developed neurofibrillary tangle pathology, tau expression was reduced by administration of doxycycline. Mouse behavioral phenotypes improved even while NFTs continued to grow. This was definitive evidence that the tangles themselves are not the prion or the “protein*”. With that prefacing, she launched into recent unpublished work on tau.
She later spent some time discussing the search for the species of Aβ responsible for neuronal impairment [Lesne 2006], and the development of antibodies against soluble oligomers [Kayed 2003]. Then she went into recent data, some of which are in press, on characterizing distinct species of Aβ oligomers.