Prions and prion diseases. Bi156 lecture, 2/13/12

Transcription

1 Prions and prion diseases Bi156 lecture, 2/13/12

2 Transmissible spongiform encephalopathies: history TSEs are uniformly fatal diseases in which the brain becomes full of holes, exhibiting a spongelike appearance in autopsy slides. They were originally called slow virus diseases because of their very long incubation times. The first human TSE to be characterized was kuru (meaning trembling in the language of the Fore people of New Guinea).

3 Kuru Kuru was a mysterious condition that affected a large fraction of the Fore population in the mid-20th century, and was the most common cause of death. Gajdusek and colleagues determined that kuru was caused by ingestion of human brains. The brains of dead Fore and of enemy tribesmen were packed into bamboo cylinders, steamed, and eaten, as was the rest of the meat. Kuru symptoms appeared years after ingestion of these brains. Gadjusek showed that kuru was infectious by transmitting it to chimpanzees.

4 Scrapie Scrapie (so-called because sick sheep scrape themselves against posts), a disease with kuru-like pathology, is endemic in sheep from some areas. Scrapie could be transmitted to hamsters by intracerebral or IV (less efficient) inoculation of brain extract from infected sheep. This allowed study of the agent, since the hamsters could exhibit pathology in 60 days.

5 Mad cow disease Creutzfeldt-Jakob disease (CJD) is a rare condition that usually appears spontaneously, but can also be transmitted by infected surgical instruments and by growth hormone purified from human pituitaries. Kuru presumably originated in New Guinea with the consumption of brain from an individual with CJD. In the 1980s, BSE (aka mad cow disease) appeared in cows in England that had been fed meat and bone meal made from sheep, some of which must have been scrapie-infected. A few hundred people who ate BSE-infected British beef developed a CJDlike condition, but one which developed more rapidly than normal CJD; this was called variant (v)cjd. This mad cow disease epidemic appears to be abating, suggesting that few new cases will be reported now that sheep and cow products are no longer fed to cows. However, practices persist in the meat industry which could still allow BSE transmission in the future/ Also, wild eld and deer on deer farms are subject to chronic wasting disease, which is like BSE; so one should be cautious about eating venison.

6 Characterization of the scrapie agent Studies of scrapie transmission to hamsters showed that the agent is resistant to UV, X-rays, and boiling (all treatments that would inactivate nucleic acids). Mad cow disease was transmitted by boiled, rendered sheep and cow byproducts. It passes through filters that trap viruses. This led to Griffith s protein-only hypothesis, in which it was proposed that the scrapie agent lacked nucleic acid; which raised the question of how such an agent could be infectious. Prusiner and colleagues purified the scrapie agent and showed that it consisted of aggregates of a single protein, called PrP.

7 PrP aggregates in the brain

8 Purification of scrapie agent The key observation necessary for isolation of agent was that it is resistant to proteinase K (compare + lanes; left is scrapie brain, right is normal brain) under conditions where all other proteins in brain homogenate are digested to completion.

9 The prion hypothesis Sequencing of the scrapie protein PrP showed that it is encoded by the host genome. PrP is a ~230 amino acid cell-surface protein, expressed in many tissues, which is attached to the outside of the cell by a lipid (glycosyl-phosphatidylinositol (GPI)) linkage. Scrapie PrP (PrP-sc) (also called PrP-res) from brains of affected hamsters is insoluble and protease-resistant. PrP from normal hamsters (PrP-c) is protease-sensitive. The prion hypothesis is that PrP-sc is an infectious agent that induces conversion of host PrP-c to the pathological PrP-sc conformation. In this manner, scrapie agent can be said to replicate despite a lack of any genetic material.

10 Arguments against the prion hypothesis Although Prusiner received the Nobel Prize for his prion work, many investigators disbelieved the hypothesis until the 1990s (and a few still do). Two of the major problems with the hypothesis: Although the agent did not seem to contain nucleic acid, there are >10 5 PrP molecules per infectious unit; so PrP is not very infectious, and scrapie agent preps could be contaminated with small amounts of virus. There are >20 different strains of scrapie, with different incubation times and pathologies; this would seem to be inconsistent with a host-encoded agent.

11 Evidence relevant to the prion hypothesis: human genetics Rare TSE-like conditions exist in humans that are genetically transmitted in an autosomal dominant manner. These include familial (f)cjd, Gerstmann-Straussler syndrome (GSS), and familial fatal insomnia (FFI). Sequencing of the PrP gene in these families showed that fcjd, GSS, and FFI are each associated with characteristic missense mutations in the PrP coding sequence. Transgenic mice expressing mouse PrP with a GSS mutation develop spongiform pathology. The brains of these mice can transmit disease.

12 Evidence relevant to the prion hypothesis: the species barrier Infection of mice with hamster scrapie produces disease with a very long incubation time (>200 days). Similarly, mouse scrapie has a long incubation time in hamsters. However, transgenic mice expressing the hamster PrP gene display a short incubation time when infected by hamster scrapie; and agent from brains of these mice kills hamsters as fast as hamster scrapie. Normal mice cannot be infected with human CJD, but transgenic mice expressing human PrP can. Thus, the PrP genotype of the host influences the nature of the infectious agent it produces: not a virus-like behavior.

13 The PrP gene is required for disease. Knock-out mice lacking PrP expression are viable. They are totally resistant to scrapie infection, showing that if a virus is present it requires host PrP for its propagation. If you make a cerebral graft from a wt mouse into a PrP- KO, the graft can be infected with scrapie, but pathology does not extend beyond the graft border. Normal mice can be infected with scrapie intraperitoneally, but grafts in these KO mice can only be infected by direct intracerebral injection. This implies that the scrapie agent can only travel by direct cell-cell contact. Recent work suggests that ingested prion agent travels from the intestine to the spleen and other lymphoid organs, then along sympathetic nerves to the brain.

14 Evidence relevant to the prion hypothesis: strains Strain differences have been rationalized by hypothesizing that PrP-sc can exist in many different conformations, and that each of these seeds conversion of PrP-c to that same PrP-sc conformation. This seems very unlikely, but is supported by experiments with transgenic mice bearing human prion disease mutations.

15 Strain transmission experiment FFI is caused by the D178M mutation, and PK digestion of PrP-sc from FFI brains generates a 19 kd resistant product. GSS is caused by the E200K mutation, and PK digestion of PrP-sc from GSS brain generates a 21 kd resistant product. FFI and GSS brain extracts can both transmit disease to transgenic mice carrying wild-type human PrP sequences. Incubation times and pathology in these mice differ between the diseases.

16 Strain transmission experiment, contd. Brain extract from the FFI or GSS-infected mice can be used for another round of infection, and pathology and incubation time characteristics are maintained in the second round. Digestion of PrP-sc from the brains of first or second cycle FFI-infected mice generates the 19 kd resistant product. Digestion of PrP-sc from brains of first or second cycle GSS-infected mice generates the 21 kd resistant product. Thus, the FFI and GSS strains breed true even when they are generated by the same hosts.

17 Structural differences between PrP-c and PrP-sc PrP-sc structure cannot be determined directly since it is an aggregate and cannot be crystallized. Analysis by circular dichroism and other techniques shows that PrP-sc is made up of beta-sheets.

18 PrP-sc is an amyloid PrP-sc forms amyloids (fibers that bind specific dyes such as Congo Red) like those seen in AD. Amyloid fibers are made of stacked beta-sheets. Amyloids can be made from many different protein sequences.

19 Hypotheses for PrP-c to PrP-sc conversion Since many prion strains exist, there should be many different PrP-sc structures, each of which can catalyze conversion of PrP-c to the same structure as itself. The seeded nucleation model hypothesizes that flipping into a (single?) prion state might occur more often, but the sc monomer hardly ever aggregates to form a seed. Once a seed forms, it recruits additional sc monomers. In this model, the infectious prion is necessarily an sc aggregate rather than a monomer. A problem with seeded nucleation: how can it explain transmission between cells, if aggregates are not present on cell surfaces?

20 Models for conversion mechanisms

21 Seeded nucleation can explain formation of distinct prion strains

22 Amplification of prions in vitro by PMCA (protein misfolding cyclic amplification)

23 PMCA shows that PrP is the only component of infected brain that is needed PMCA is a way of greatly increasing the efficiency of seeded nucleation. It starts with a seed of scrapie brain extract. PMCA has been performed for enough cycles to show that none of the original prion remains in the infectious prep. The original version of PMCA required crude brain material, however, so it could not show that PrP-sc is the only component required for infectivity.

24 Creation of the prion conformation in vitro Since amyloids can be readily made from denatured proteins in vitro, if PrP-sc (Prp-res) is an amyloid it should be possible to generate infectious PrP-sc from recombinant protein, thus providing the final proof of the prion hypothesis. However, the nature of the infectious species is unclear; it is probably a transient intermediate rather than a large amyloid and this could not be made in vitro, although many people tried, both before and after PMCA was developed. In 2010, it was finally reported that transmissible disease could be created starting with only recombinant or synthetic materials.

25 An infectious recombinant prion The key ingredients are: recombinant PrP from E. coli, a synthetic anionic phospholipid, and RNA. Applying PMCA to this mixture generates an aggregate after 18 cycles, and this causes transmissible disease in mice.

26 How do prion aggregates move within cells? This figure suggests that PrP-res aggregate can convert PrP-c on the cell surface or in endosomes. It also shows a model for toxicity in which aggregate affects protein folding in the ER.

27 How do prion aggregates spread from one cell to the next? Two hypothetical mechanisms: Exosomes (small membranous secreted particles) and TNTs (tunneling nanotubes). Once in vesicles within the recipient cell, PrP-res can convert endogenous PrP-c to aggregates that can be exported from that cell in turn.

28 How is pathology generated in prion diseases? Although PrP-sc aggregates form amyloid fibers, there is no evidence that these are actually the agents which kill brain cells. Furthermore, some prion conditions are not associated with accumulation of PrP-sc. There are many models for how PrP-sc accumulation leads to death of neurons. One model is based on the fact that PrP-c can exist in three forms, one of which may be toxic.

29 PrP isoforms PrP isoforms The normal cell-surface form, which translocates all the way through the endoplasmic reticulum (ER) membrane and then becomes linked to it by lipid, is called PrP-sec. There are two other forms, however: Ntm-PrP, which is transmembrane, with the N-terminus inside the ER vesicle, and Ctm-PrP, also transmembrane, with the C terminus inside. In vitro translation with crude (ER) membranes generates all three forms, but translation with purified lipid vesicles produces only Ctm-PrP. This implies that generation of PrP-sec and Ntm requires trans-acting factors missing from the purified lipid vesicles.

30 Ctm-PrP is associated with spongiform pathology Mutations in PrP can change the ratio of Ctm-PrP to PrP-sec generated by in vitro translation with crude membranes. High-level expression of Ctm-PrP favoring mutations in mice produces spontaneous disease, but there is no generation of PrP-sc and the brain lysate is not infective. This suggests that disease is produced by accumulation of Ctm-PrP. In TSEs where PrP-sc is present, it facilitates the generation of Ctm-PrP at the expense of the other forms. A problem with this model: Ctm-PrP has never been detected in vivo.

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32 Studying prions in yeast The characteristics of scrapie and PrP makes it difficult to rapidly characterize the biochemical and genetic properties of prions, because disease generation requires a long time and the conditions within the animal cannot be easily monitored. Studies in yeast show that protein-based genetic elements are not unique to PrP. Study of these prion proteins (Sup35p and Ure2p) has proven that proteins can transmit information across generations. It has also provided insights into the nature of prion strains, mechanisms of prion propagation, and roles of prions in normal cell physiology.

33 Yeast prions: [PSI+] Sup35 is a translation termination factor. In normal (psi-) cells, Sup35 is soluble and causes efficient termination of translation at stop codons. In PSI+ cells, Sup35 is aggregated, and the reduction in functional, soluble Sup35 causes read-through of certain stop codons. This allows for selection of the PSI+ state, by using stop codon mutations in a selectable gene such as ADE1. The PSI+ state is passed down to descendants and can be transmitted to other yeast by cytoduction (transfer of cytoplasm without genetic material). Thus, PSI+ is a heritable state that is transmitted by a protein.

34 Sup35p and Ure2p: prion phenotypes

35 Characteristics of yeast prions The prion state is not simply aggregation of a protein. For a protein to function as a prion, the aggregate and soluble states must be interconvertible. The aggregate must be able to convert soluble protein to the same structure as the protein in the aggregate. The aggregate must pass down these characteristics to daughter cells.

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37 Yeast prions form amyloids Sup35 and other yeast prions form fibers in vitro that are congophilic (bind Congo Red dye), which is a characteristic of mammalian amyloid fibers. EM images of these fibers appear similar to those of PrP-sc amyloid fibers.

38 Sup35 fibers can seed formation of new fibers through nucleated polymerization. Depending on ionic conditions, these fibers can take on a number of different forms. These forms are inheritable in vitro, in that each seeds formation of new fibers with the same form.

39 Strain phenotypes in yeast and mice Different strains of PrP-sc prions show differing sensitivities to protein denaturants and cause different pathologies. Similarly, strains of SUP35 prions with different thermal sensitivities can be produced in vitro using different seeds; in vivo these generate different color phenotypes.

40 Direct transmission of the prion state by protein made in vitro. Introduction of fibers made in vitro into psi- yeast converts them to PSI+, proving that the in vitro generated fiber is the same as the in vivo inherited genetic element.

41 Summary of parallels between yeast and mammalian prions

42 Generalization of the prion model to aggregateassociated neurodegenerative diseases Recent data indicate that Abeta, SAA, synuclein, tau, and SOD aggregates can transfer between cells. This suggests that seeded nucleation is not limited to amyloid aggregates.

43 Model for transfer of amyloids between cells

44 Spreading of ALS-linked aggregates

45 Designer prions A prion-forming domain can be attached to another selectable function to form a novel prion. Joining the Sup35 prion domain to the DNA-binding domain of a nuclear hormone receptor generates a protein that switches on expression of a reporter gene (in this case beta-galactosidase, which makes the yeast blue on an indicator plate). However, when this protein, NMGR, is incorporated into an aggregate it is nonfunctional so the yeast turn white. White and blue states are heritable but can interconvert, demonstrating that the new prion protein can exist in two stable and mutually exclusive states.

46 NMGR prion color phenotypes

47 Characteristics of yeast prion proteins Yeast prions contain regions rich in Gln (Q) and Asn (N) amino acid residues. These regions also typically contain short repeated amino acid sequences. The prion-forming domain is separate from the functional domain.

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49 Prion-forming domains are unusually rich in Gln and Asn. The Q/N rich nature of prion-forming domains suggested that new prions might be found by identifying Q/N rich sequence blocks in other proteins, then testing their ability to form prions by linking them to Sup35.

50 Identification of new yeast prion genes This method identified two new prions in yeast, New1p and Rnq1p. Their genes are not essential, so aggregation of these proteins, which can occur in normal cells, has no phenotypic consequences. When New1p and Rnq1p Q/N rich domains are linked to Sup35, heritable prions are produced. These prions do not convert Sup35 with its normal prionforming domain to a prion state. GFP-tagged New1p and Rnq1p aggregate, but these aggregates segregate away from Sup35p aggregates. Thus, these three prions can propagate independently in the same cell.

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52 Reversing the prion state PSI+ cells in which Sup35p is aggregated can be converted to psi- cells with soluble Sup35p by growing them with low concentrations of a protein denaturant, guanidine hydrochloride. Aggregates can also be disassembled and PSI+ converted to psi- by overproduction of the chaperone Hsp104p. Curiously, deletion of the Hsp104 gene also prevents prion propagation.

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55 Prions in normal cell physiology In the fungus Podospora, heterokaryon formation is only allowed between colonies that have identical alleles at a locus called het-s. [Het-s] can fuse only with the same strain, but [Het-s*] can fuse with strains bearing other alleles as well. [Het-s] is a prion, and [Het-s*] is the absence of this prion. Thus, in Podospora the prion facilitates normal function of the organism.

56 Evolutionary benefits of PSI+? The PSI+ state could be beneficial to yeast by allowing them to adapt rapidly to new conditions by reading through stop codons, thus making a gallery of proteins with C- terminal extensions. If one of these new proteins is useful and allows the yeast to survive, later mutation of the stop codon would allow this new protein to become part of the normal proteome. In this manner, PSI+-driven readthrough could facilitate evolution by generating a mutator -like response to stress conditions.

57 Model for PSI+ as a facilitator of genetic variation Readthrough of stop codons can generate new protein domains, but can also merge genes and destabilize RNAs, thus turning off genes.

58 Are prions used as stable state switches? Eukaryotic proteomes contain many Q/N blocks (hundreds in Drosophila). If some of these proteins are prions, what could prion conversion be used for in normal animals? Prion conversion would allow a protein species to be locked into a state beyond the lifetime of any individual protein molecule. This could permanently switch genes on or off within a cell lineage, since the prion state could survive cell division. It could also lock in a local translational state at a synapse, and this mechanism has been proposed to be a mechanism for maintenance of long-term synaptic states.

59 Reviews *Getting a Grip on Prions: Oligomers, Amyloids, and Pathological Membrane Interactions. B. Caughey et al., Ann. Rev. Biochem. 78, (2009). *The Seeds of Neurodegeneration: Prion-like Spreading in ALS. M. Polymenidou and D. W. Cleveland, Cell 147, 498 (2011). The prion s elusive reason for being. (2008) Aguzzi, A., et al., Ann. Rev. Neurosci. 31, Protein Homeostasis and the Phenotypic Manifestation of Genetic Diversity: Principles and Mechanisms. D. F. Jarosz et al., Ann. Rev. Genet. 44, (2010). *Required reading

60 Papers for student presentations Opposing Effects of Glutamine and Asparagine Govern Prion Formation by Intrinsically Disordered Proteins. R. Halfmann et al., Mol. Cell 43, (2011). Generating a Prion with Bacterially Expressed Recombinant Prion Protein. F. Wang et al., Science 327, 1132 (2010). Conversion of a yeast prion protein to an infectious form in bacteria. S. J. Garrity et al., PNAS 107, (2010).