Table of Contents

Volume 6, Issue 3

  July/August 2012

REVIEWS

Patterns of [PSI⁺] aggregation allow insights into cellular organization of yeast prion aggregates

Jens Tyedmers
http://dx.doi.org/10.4161/pri.18986
Abstract | Full Text | PDF

The yeast prion phenomenon is very widespread and mounting evidence suggests that it has an impact on cellular regulatory mechanisms related to phenotypic responses to changing environments. Studying the aggregation patterns of prion amyloids during different stages of the prion life cycle is a first key step to understand major principles of how and where cells generate, organize and turn-over prion aggregates. The induction of the [PSI⁺] state involves the actin cytoskeleton and quality control compartments such as the Insoluble Protein Deposit (IPOD). An initially unstable transitional induction state can be visualized by overexpression of the prion determinant and displays characteristic large ring- and ribbon-shaped aggregates consisting of poorly fragmented bundles of very long prion fibrils. In the mature prion state, the aggregation pattern is characterized by highly fragmented, shorter prion fibrils that form aggregates, which can be visualized through tagging with fluorescent proteins. The number of aggregates formed varies, ranging from a single large aggregate at the IPOD to multiple smaller ones, depending on several parameters discussed. Aggregate units below the resolution of light microscopy that are detectable by fluorescence correlation spectroscopy are in equilibrium with larger aggregates in this stage and can mediate faithful inheritance of the prion state. Loss of the prion state is often characterized by reduced fragmentation of prion fibrils and fewer, larger aggregates.

Probing the role of structural features of mouse PrP in yeast by expression as Sup35-PrP fusions

Lyne Jossé, Ricardo Marchante, Jo Zenthon, Tobias von der Haar and Mick F. Tuite
Abstract | Full Text | PDF

The yeast Saccharomyces cerevisiae is a tractable model organism in which both to explore the molecular mechanisms underlying the generation of disease-associated protein misfolding and to map the cellular responses to potentially toxic misfolded proteins. Specific targets have included proteins which in certain disease states form amyloids and lead to neurodegeneration. Such studies are greatly facilitated by the extensive ‘toolbox’ available to the yeast researcher that provides a range of cell engineering options. Consequently, a number of assays at the cell and molecular level have been set up to report on specific protein misfolding events associated with endogenous or heterologous proteins. One major target is the mammalian prion protein PrP because we know little about what specific sequence and/or structural feature(s) of PrP are important for its conversion to the infectious prion form, PrP^Sc. Here, using a study of the expression in yeast of fusion proteins comprising the yeast prion protein Sup35 fused to various regions of mouse PrP protein, we show how PrP sequences can direct the formation of non-transmissible amyloids and focus in particular on the role of the mouse octarepeat region. Through this study we illustrate the benefits and limitations of yeast-based models for protein misfolding disorders.




EXTRA VIEWS

Domain swapping and amyloid fibril conformation

Patrick C.A. van der Wel
Abstract | Full Text | PDF

For several different proteins an apparent correlation has been observed between the propensity for dimerization by domain-swapping and the ability to aggregate into amyloid-like fibrils. Examples include the disease-related proteins β₂-microglobulin and transthyretin. This has led to proposals that the amyloid-formation pathway may feature extensive domain swapping. One possible consequence of such an aggregation pathway is that the resulting fibrils would incorporate structural elements that resemble the domain-swapped forms of the protein and, thus, reflect certain native-like structures or domain-interactions. In magic angle spinning solid-state NMR-based and other structural studies of such amyloid fibrils, it appears that many of these proteins form fibrils that are not native-like. Several fibrils, instead, have an in-register, parallel conformation, which is a common amyloid structural motif and is seen, for instance, in various prion fibrils. Such a lack of native structure in the fibrils suggests that the apparent connection between domain-swapping ability and amyloid-formation may be more subtle or complex than may be presumed at first glance.

Regulation of amyloid-β production by the prion protein

Heledd H. Griffiths, Isobel J. Whitehouse and Nigel M. Hooper
Abstract | Full Text | PDF

Alzheimer disease (AD) is characterized by the amyloidogenic processing of the amyloid precursor protein (APP), culminating in the accumulation of amyloid-β peptides in the brain. The enzymatic action of the β-secretase, BACE1 is the rate-limiting step in this amyloidogenic processing of APP. BACE1 cleavage of wild-type APP (APP_WT) is inhibited by the cellular prion protein (PrP^C). Our recent study has revealed the molecular and cellular mechanisms behind this observation by showing that PrP^C directly interacts with the pro-domain of BACE1 in the trans-Golgi network (TGN), decreasing the amount of BACE1 at the cell surface and in endosomes where it cleaves APP_WT, while increasing BACE1 in the TGN where it preferentially cleaves APP with the Swedish mutation (APP_Swe). PrP^C deletion in transgenic mice expressing the Swedish and Indiana familial mutations (APP_Swe,Ind) failed to affect amyloid-β accumulation, which is explained by the differential subcellular sites of action of BACE1 toward APP_WT and APP_Swe. This, together with our observation that PrP^C is reduced in sporadic but not familial AD brain, suggests that PrP^C plays a key protective role against sporadic AD. It also highlights the need for an APP_WT transgenic mouse model to understand the molecular and cellular mechanisms underlying sporadic AD.

Is tau ready for admission to the prion club?

Garth F. Hall and Brian A. Patuto
Abstract | PDF

Aggregation-prone proteins associated with neurodegenerative disease such as alpha synuclein and beta amyloid now appear to share key prion-like features with mammalian prion protein, such as the ability to recruit normal proteins to aggregates and to translocate between neurons. These features may shed light on the genesis of stereotyped lesion development patterns in conditions such as Alzheimer disease and Lewy Body dementia. We discuss the qualifications of tau protein as a possible “prionoid” mediator of lesion spread based on recent characterizations of the secretion, uptake and transneuronal transfer of human tau isoforms in a variety of tauopathy models and in human patients. In particular, we consider (1) the possibility that prionoid behavior of misprocessed tau in neurodegenerative disease may involve other aggregation-prone proteins, including PrP itself and (2) whether “prionlike” tau lesion propagation might include mechanisms other than protein-protein templating.

A new perspective on Hsp104-mediated propagation and curing of the yeast prion [PSI⁺]

Christopher W. Helsen and John R. Glover
Abstract | PDF

Most prions in yeast form amyloid fibrils that must be severed by the protein disaggregase Hsp104 to be propagated and transmitted efficiently to newly formed buds. Only one yeast prion, [PSI⁺], is cured by Hsp104 overexpression. We investigated the interaction between Hsp104 and Sup35, the priongenic protein in yeast that forms the [PSI⁺] prion.1 We found that a 20-amino acid segment within the highly-charged, unstructured middle domain of Sup35 contributes to the physical interaction between the middle domain and Hsp104. When this segment was deleted from Sup35, the efficiency of [PSI⁺] severing was substantially reduced resulting in larger Sup35 particles and weakening of the [PSI⁺] phenotype. Furthermore, [PSI⁺] in these cells was completely resistant to Hsp104 curing. The affinity of Hsp104 was considerably weaker than that of model Hsp104-binding proteins and peptides and implying that Sup35 prions are not ideal substrates for Hsp104-mediated remodeling. In light of this finding, we present a modified model of Hsp104-mediated [PSI⁺] propagation and curing that requires only partial remodeling of Sup35 assembled into amyloid fibrils.

Hul5 ubiquitin ligase: Good riddance to bad proteins

Nancy N. Fang and Thibault Mayor
Abstract | PDF

Failure to eliminate abnormal proteins in the cell is associated with numerous aggregation diseases. Misfolded proteins are normally detected by protein quality control and either refolded or eliminated. The ubiquitin-proteasome system is a major pathway that degrades these unwanted proteins. Ubiquitin ligases are central to these degradation pathways as they recognize aberrant proteins and covalently attach a polyubiquitin chain to target them to the proteasome. We discovered that the Hul5 ubiquitin ligase is a major player in a novel protein quality control pathway that targets cytosolic misfolded proteins. Hul5 is required for the maintenance of cell fitness and the increased ubiquitination of low solubility proteins after heat-shock in yeast cells. We identified several low-solubility substrates of Hul5, including the prion-like protein Pin3. It is now apparent that in the cytoplasm, misfolded proteins can be targeted by multiple degradation pathways. In this review, we discuss how the Hul5 protein quality control pathway may specifically target low solubility cytosolic proteins in the cell.

Unraveling the neuroprotective mechanisms of PrP^C in excitotoxicity

Franc Llorens and José Antonio del Río
Abstract | Full Text | PDF

Knowledge of the natural roles of cellular prion protein (PrP^C) is essential to an understanding of the molecular basis of prion pathologies. This GPI-anchored protein has been described in synaptic contacts, and loss of its synaptic function in complex systems may contribute to the synaptic loss and neuronal degeneration observed in prionopathy. In addition, Prnp knockout mice show enhanced susceptibility to several excitotoxic insults, GABA_A receptor-mediated fast inhibition was weakened, LTP was modified and cellular stress increased. Although little is known about how PrP^C exerts its function at the synapse or the downstream events leading to PrP^C-mediated neuroprotection against excitotoxic insults, PrP^C has recently been reported to interact with two glutamate receptor subunits (NR2D and GluR6/7). In both cases the presence of PrP^C blocks the neurotoxicity induced by NMDA and Kainate respectively. Furthermore, signals for seizure and neuronal cell death in response to Kainate in Prnp knockout mouse are associated with JNK3 activity, through enhancing the interaction of GluR6 with PSD-95. In combination with previous data, these results shed light on the molecular mechanisms behind the role of PrP^C in excitotoxicity. Future experimental approaches are suggested and discussed.

Open Access Article

Genesis of tramsmissible protein states via deformed templating

Natallia Makarava and Ilia V. Baskakov
Abstract | Full Text | PDF

Prion replication occurs via a template-assisted mechanism, which postulates that the folding pattern of a newly recruited polypeptide chain accurately reproduces that of a template. The concept of prion-like template-assisted propagation of an abnormal protein conformation has been expanded to amyloidogenic proteins associated with Alzheimer, Parkinson, Huntington diseases, amyotrophic lateral sclerosis and others. Recent studies demonstrated that authentic PrP^Sc and transmissible prion disease could be generated in wild type animals by inoculation of recombinant prion protein amyloid fibrils, which are structurally different from PrP^Sc and lack any detectable PrP^Sc particles. Here we discuss a new replication mechanism designated as “deformed templating,” according to which fibrils with one cross-β folding pattern can seed formation of fibrils or particles with a fundamentally different cross-β folding pattern. Transformation of cross-β folding pattern via deformed templating provides a mechanistic explanation behind genesis of transmissible protein states induced by amyloid fibrils that are considered to be non-infectious. We postulate that deformed templating is responsible for generating conformationally diverse amyloid populations, from which conformers that are fit to replicate in a particular cellular environment are selected. We propose that deformed templating represents an essential step in the evolution of transmissible protein states.




SHORT COMMUNICATION

Polymorphisms and variants in the prion protein sequence of European Moose (Alces alces), Reindeer (Rangifer tarandus), Roe Deer (Capreolus capreolus) and Fallow Deer (Dama dama) in Scandinavia

Lotta Wik, Sofia Mikko, Mikael Klingeborn, Margareta Stéen, Magnus Simonsson and Tommy Linné
Abstract | Full Text | PDF

The prion protein (PrP) sequence of European moose, reindeer, roe deer and fallow deer in Scandinavia has high homology to the PrP sequence of North American cervids. Variants in the European moose PrP sequence were found at amino acid position 109 as K or Q. The 109Q variant is unique in the PrP sequence of vertebrates. During the 1980s a wasting syndrome in Swedish moose, Moose Wasting Syndrome (MWS), was described. SNP analysis demonstrated a difference in the observed genotype proportions of the heterozygous Q/K and homozygous Q/Q variants in the MWS animals compared with the healthy animals. In MWS moose the allele frequencies for 109K and 109Q were 0.73 and 0.27, respectively, and for healthy animals 0.69 and 0.31. Both alleles were seen as heterozygotes and homozygotes. In reindeer, PrP sequence variation was demonstrated at codon 176 as D or N and codon 225 as S or Y. The PrP sequences in roe deer and fallow deer were identical with published GenBank sequences.




RESEARCH PAPERS

Open Access Article

Phenotypic characterization of cells participating in transport of prion protein aggregates across the intestinal mucosa of sheep

Caroline Piercey Åkesson, Charles McL. Press, Michael A. Tranulis, Martin Jeffrey, Mona Aleksandersen, Thor Landsverk and Arild Espenes
Abstract | Full Text | PDF

The oral route is considered to be the main entry site of several transmissible spongiform encephalopathies or prion diseases of animals and man. Following natural and experimental oral exposure to scrapie, sheep first accumulate disease associated prion protein (PrP^d) in Peyer’s patch (PP) lymphoid follicles. In this study, recombinant ovine prion protein (rPrP) was inoculated into gut loops of young lambs and the transportation across the intestinal wall studied. In particular, the immunohistochemical phenotypes of cells bearing the inoculated prion protein were investigated. The rPrP was shown to be transported across the villi of the gut, into the lacteals and submucosal lymphatics, mimicking the transport route of PrP^d from scrapie brain inoculum observed in a previous intestinal loop experiment. The cells bearing the inoculated rPrP were mainly mononuclear cells, and multicolor immunofluorescence procedures were used to show that the rPrP bearing cells were professional antigen presenting cells expressing Major histocompatibility complex II (MHCII). In addition, the rPrP bearing cells labeled with CD205, CD11b and the macrophage marker CD68, and not with the dendritic cell markers CD11c and CD209. Others have reported that cells expressing CD205 and CD11b in the absence of CD11c have been shown to induce T cell tolerance or regulatory T cells. Based on this association, it was speculated that the rPrP and by extension PrP^d and scrapie infective material may exploit the physiological process of macromolecular uptake across the gut, and that this route of entry may have implications for immune surveillance.

Correlation analysis for the incubation period of prion disease

Se-Eun Bae, Sunghoon Jung, Ha-Yeon Kim and Hyeon S. Son
Abstract | Full Text | PDF

Previous studies have shown that genetic quantitative trait loci (QTL), strain barriers, inoculation dose and inoculation method modulate the incubation period of prion diseases. We examined the relationship between a diverse set of physical, genetic and immunological characteristics and the incubation period of prion disease using correlation analyses. We found that incubation period was highly correlated with brain weight. In addition, mean corpuscular volume and cell size were strongly correlated with incubation period, indicating that the physical magnitude of prion-infected organs or individual cells may be important in determining the incubation period. Given the same prion inoculation dose, animals with a lower brain weight, mean corpuscular volume or cell size may experience more virulent disease, as the effective concentration of abnormal prion, which might regulate the attachment rate of prions to aggregates, is increased with smaller capacity of brains and cells. This is partly consistent with previous theoretical modeling. The strong correlations between incubation period and physical properties of the brain and cells in this study suggest that the mechanism underlying prion disease pathology may be physical, indicating that the incubation process is governed by simple chemical stoichiometry.

Gene expression alterations in Rocky Mountain elk infected with chronic wasting disease

Urmila Basu, Luciane M. Almeida, Sandor Dudas, Catherine E. Graham, Stefanie Czub, Stephen S. Moore and Le Lou Guan
Abstract | PDF

Chronic wasting disease (CWD) is an invariably fatal neurologic disease that naturally infects mule deer, white tailed deer and elk. The understanding of CWD neurodegeneration at a molecular level is very limited. In this study, microarray analysis was performed to determine changes in the gene expression profiles in six different tissues including brain, midbrain, thalamus, spleen, RPLN and tonsil of CWD-infected elk in comparison to non-infected healthy elk, using 24,000 bovine specific oligo probes. In total, 329 genes were found to be differentially expressed (> 2.0-fold) between CWD negative and positive brain tissues, with 132 genes up-regulated and 197 genes down-regulated. There were 249 DE genes in the spleen (168 up- and 81 down-regulated), 30 DE genes in the retropharyngeal lymph node (RPLN) (18 up- and 12 down-regulated), and 55 DE genes in the tonsil (21 up- and 34 down-regulated). Using Gene Ontology (GO), the DE genes were assigned to functional groups associated with cellular process, biological regulation, metabolic process, and regulation of biological process. For all brain tissues, the highest ranking networks for DE genes identified by Ingenuity Pathway Analysis (IPA) were associated with neurological disease, cell morphology, cellular assembly and organization. Quantitative real-time PCR (qRT-PCR) validated the expression of DE genes primarily involved in different regulatory pathways, including neuronal signaling and synapse function, calcium signaling, apoptosis and cell death and immune cell trafficking and inflammatory response. This is the first study to evaluate altered gene expression in multiple organs including brain from orally infected elk and the results will improve our understanding of CWD neurodegeneration at the molecular level.

Temperature influences the interaction of ruminant PrP^TSE with soil

Ben C. Maddison, Jonathan P. Owen, Maged A. Taema, George Shaw and Kevin C. Gough
Abstract | Full Text | PDF

Ovine scrapie and cervid chronic wasting disease can be transmitted in the absence of animal-to-animal contact, and environmental reservoirs of infectivity have been implicated in their spread and persistence. Investigating environmental factors that influence the interaction of disease-associated PrP with soils is imperative to understanding what is likely to be the complex role of soil in disease transmission. Here, we describe the effects of soil temperature on the binding/desorption and persistence of both ovine scrapie- and bovine BSE-PrP^TSE. Binding of PrP^TSE to a sandy loam soil at temperatures of 4°C, 8–12°C and 25–30°C demonstrated that an increase in temperature resulted in (1) a decrease in the amount of PrP^TSE recovered after 24 h of interaction with soil, (2) an increase in the amount of N-terminal cleavage of the prion protein over 11 d and (3) a decrease in the persistence of PrP^TSE on soil over an 18 mo period.

The role of Bax and caspase-3 in doppel-induced apoptosis of cerebellar granule cells

Alessandro Didonna, Joshua Sussman, Federico Benetti and Giuseppe Legname
Abstract | PDF

Doppel (Dpl) protein is a paralog of the prion protein (PrP) that shares 25% sequence similarity with the C-terminus of PrP, a common N-glycosylation site and a C-terminal signal peptide for attachment of a glycosylphophatidyl inositol anchor. Whereas PrPC is highly expressed in the central nervous system (CNS), Dpl is detected mostly in testes and its ectopic expression in the CNS leads to ataxia as well as Purkinje and granule cell degeneration in the cerebellum. The mechanism through which Dpl induces neurotoxicity is still debated. In the present work, primary neuronal cultures derived from postnatal cerebellar granule cells of wild-type and PrP-knockout FVB mice were used in order to investigate the molecular events that occur upon exposure to Dpl. Treatment of cultured cerebellar neurons with recombinant Dpl produced apoptosis that could be prevented by PrP co-incubation. When primary neuronal cultures from Bax-deficient mice were incubated with Dpl, no apoptosis was observed, suggesting an important role of Bax in triggering neurodegeneration. Similarly, cell survival increased when recDpl-treated cells were incubated with an inhibitor of caspase-3, which mediates apoptosis in mammalian cells. Together, our findings raise the possibility that Bax and caspase-3 feature in Dpl-mediated apoptosis.