Wild-type Prion Protein Research Articles

A Nine Amino Acid Domain Is Essential for Mutant Prion Protein Toxicity

Transgenic mice expressing prion protein (PrP) molecules with several different internal deletions display spontaneous neurodegenerative phenotypes that can be dose-dependently suppressed by coexpression of wild-type PrP. Each of these deletions, including the largest one (Δ32-134), retains 9 aa immediately following the signal peptide cleavage site (residues 23-31; KKRPKPGGW). These residues have been implicated in several biological functions of PrP, including endocytic trafficking and binding of glycosaminoglycans. We report here on our experiments to test the role of this domain in the toxicity of deleted forms of PrP. We find that transgenic mice expressing Δ23-134 PrP display no clinical symptoms or neuropathology, in contrast to mice expressing Δ32-134 PrP, suggesting that residues 23-31 are essential for the toxic phenotype. Using a newly developed cell culture assay, we narrow the essential region to amino acids 23-26, and we show that mutant PrP toxicity is not related to the role of the N-terminal residues in endocytosis or binding to endogenous glycosaminoglycans. However, we find that mutant PrP toxicity is potently inhibited by application of exogenous glycosaminoglycans, suggesting that the latter molecules block an essential interaction between the N terminus of PrP and a membrane-associated target site. Our results demonstrate that a short segment containing positively charged amino acids at the N terminus of PrP plays an essential role in mediating PrP-related neurotoxicity. This finding identifies a protein domain that may serve as a drug target for amelioration of prion neurotoxicity.

Introduction of glutamines into the B2–H2 loop promotes prion protein conversion

In prion diseases cellular prion protein (PrPC) undergoes conformational transition into the β-sheet-rich form (PrPSc). PrPC consists of the disordered N-terminal part and a C-terminal globular domain containing three α-helices (H1, H2, H3) and an antiparallel beta sheet (B1, B2). B2–H2 loop, which has a focal role in the species barrier, contains the highest density of asparagine (N) and glutamine (Q) residues in the whole sequence. Q/N-rich domains are essential for the conversion of yeast prions. We investigated the role of Q/N residues in the B2–H2 loop in PrP conversion. We prepared mouse PrP mutants with increasing number of consecutive Q/N residues in the B2–H2 loop. Stability of the mutants decreased with the increasing number of inserted glutamines. In vitro conversion of mutants yielded fibrils of similar morphology as the wild-type PrP. Q/N mutants accelerated fibrillization in comparison to the wild-type PrP, with mutant containing the most glutamines having the shortest lag phase. The effect of Q/N residues was specific for the B2–H2 loop and was not due to simple increase in flexibility as the introduction of Gly-Ser or Ala residues slowed the conversion despite their decreased stability. Our results thus suggest that Q/N residues in the B2–H2 loop of PrP promote protein conversion and may represent a link to conversion of Q/N-rich prions.

Effect of Hydrophobic Mutations in the H2-H3 Subdomain of Prion Protein on Stability and Conversion In Vitro and In Vivo

Prion diseases are fatal neurodegenerative diseases, which can be acquired, sporadic or genetic, the latter being linked to mutations in the gene encoding prion protein. We have recently described the importance of subdomain separation in the conversion of prion protein (PrP). The goal of the present study was to investigate the effect of increasing the hydrophobic interactions within the H2-H3 subdomain on PrP conversion. Three hydrophobic mutations were introduced into PrP. The mutation V209I associated with human prion disease did not alter protein stability or in vitro fibrillization propensity of PrP. The designed mutations V175I and T187I on the other hand increased protein thermal stability. V175I mutant fibrillized faster than wild-type PrP. Conversion delay of T187I was slightly longer, but fluorescence intensity of amyloid specific dye thioflavin T was significantly higher. Surprisingly, cells expressing V209I variant exhibited inefficient proteinase K resistant PrP formation upon infection with 22L strain, which is in contrast to cell lines expressing wild-type, V175I and T187I mPrPs. In agreement with increased ThT fluorescence at the plateau T187I expressing cell lines accumulated an increased amount of the proteinase K-resistant prion protein. We showed that T187I induces formation of thin fibrils, which are absent from other samples. We propose that larger solvent accessibility of I187 in comparison to wild-type and other mutants may interfere with lateral annealing of filaments and may be the underlying reason for increased conversion efficiency.

Dissociation of Infectivity from Seeding Ability in Prions with Alternate Docking Mechanism

Previous studies identified two mammalian prion protein (PrP) polybasic domains that bind the disease-associated conformer PrPSc, suggesting that these domains of cellular prion protein (PrPC) serve as docking sites for PrPSc during prion propagation. To examine the role of polybasic domains in the context of full-length PrPC, we used prion proteins lacking one or both polybasic domains expressed from Chinese hamster ovary (CHO) cells as substrates in serial protein misfolding cyclic amplification (sPMCA) reactions. After ∼5 rounds of sPMCA, PrPSc molecules lacking the central polybasic domain (ΔC) were formed. Surprisingly, in contrast to wild-type prions, ΔC-PrPSc prions could bind to and induce quantitative conversion of all the polybasic domain mutant substrates into PrPSc molecules. Remarkably, ΔC-PrPSc and other polybasic domain PrPSc molecules displayed diminished or absent biological infectivity relative to wild-type PrPSc, despite their ability to seed sPMCA reactions of normal mouse brain homogenate. Thus, ΔC-PrPSc prions interact with PrPC molecules through a novel interaction mechanism, yielding an expanded substrate range and highly efficient PrPSc propagation. Furthermore, polybasic domain deficient PrPSc molecules provide the first example of dissociation between normal brain homogenate sPMCA seeding ability from biological prion infectivity. These results suggest that the propagation of PrPSc molecules may not depend on a single stereotypic mechanism, but that normal PrPC/PrPSc interaction through polybasic domains may be required to generate prion infectivity.

The Cellular Prion Protein Is Expressed in Olfactory Sensory Neurons of Adult Mice but Does Not Affect the Early Events of the Olfactory Transduction Pathway

A conformational conversion of the cellular prion protein (PrP(C)) is now recognized as the causal event of fatal neurodegenerative disorders, known as prion diseases. In spite of long-lasting efforts, however, the physiological role of PrP(C) remains unclear. It has been reported that PrP(C) is expressed in various areas of the olfactory system, including the olfactory epithelium, but its precise localization in olfactory sensory neurons (OSNs) is still debated. Here, using immunohistochemistry tools, we have reinvestigated the expression and localization of PrP(C) in the olfactory epithelium of adult congenic mice expressing different PrP(C) amounts, that is, wild-type, PrP-knockout, and transgenic PrP(C)-overexpressing animals. We found that PrP(C) was expressed in OSNs, in which, however, it was unevenly distributed, being detectable at low levels in cell bodies, dendrites and apical layer, and more abundantly in axons. We also studied the involvement of PrP(C) in the response of the olfactory epithelium to odorants, by comparing the electro-olfactograms of the 3 mouse lines subjected to different stimulation protocols. We found no significant difference between the 3 PrP genotypes, supporting previous reports that exclude a direct action of PrP(C) in the early signal transduction activity of the olfactory epithelium.

Knockdown of prion protein (PrP) by RNA interference weakens the protective activity of wild-type PrP against copper ion and antagonizes the cytotoxicity of fCJD-associated PrP mutants in cultured cells

Development of the pathogenesis of transmissible spongiform encephalopathies (TSEs) requires the presence of both the normal host prion protein (PrPC) and the abnormal pathological proteinase-K resistant isoform (PrPSc). Reduction of PrPC levels has been shown to extend survival time after prion infection. In this report, based on analysis of the known sequences of human PrP, we constructed two small interfering RNA (siRNA) duplexes targeting the segments of amino acids (aa) 108-114 (Ri2) and aa 171-177 (Ri3). Western blot analysis results revealed that these PrP-specific siRNAs could effectively knock down the levels of either endogenous PrP in human neuroblastoma SHSY-5Y cells or recombinant PrP transfected with the plasmid expressing the full-length human PrP in human embryonic kidney (HEK) 293T cells. Meanwhile, the two siRNAs also showed a significant effect on the reduction of the expression of the PrP-PG9 and PrP-PG12 familial Creutzfeldt-Jakob disease (CJD)-associated PrP mutants with four and seven extra octarepeats, in the cells transfected with the respective expression plasmids. MTT tests identified that knockdown of wild-type PrP by Ri2 and Ri3 did not change the cell growth capacities, but significantly decreased the cell resistances against the challenge of Cu2+. Co-expression of Ri2 and Ri3 partially antagonized the cytotoxicity caused by expressing PrP-PG9 and PrP-PG12 in the two cell lines. Moreover, the rescuing effectiveness of PrP siRNAs was time-related, with the more efficient antagonism of the cytotoxicity of fCJD-associated PrP mutants occurring at the early stages after transfection. The data shown here provide useful clues for seeking potential therapeutic tools for prion diseases.

Molecular Dynamics Studies on the Structural Stability of Wild-type Dog Prion Protein

Prion diseases such as Creutzfeldt-Jakob disease, variant Creutzfeldt-Jakob diseases, Gerstmann-Sträussler-Scheinker syndrome, Fatal Familial Insomnia, Kuru in humans, scrapie in sheep, bovine spongiform encephalopathy (or ‘mad-cow’ disease) and chronic wasting disease in cattle are invariably fatal and highly infectious neurodegenerative diseases affecting humans and animals. However, by now there have not been some effective therapeutic approaches to treat all these prion diseases. In 2008, canine mammals including dogs (canis familials) were the first time academically reported to be resistant to prion diseases (Vaccine 26: 2601-2614 (2008)). Thus, it is very worth studying the molecular structures of dog prion protein to obtain insights into the immunity of dogs to prion diseases. This paper studies the molecular structural dynamics of wild-type dog prion protein. The comparison analyses with rabbit prion protein show that the dog prion protein has stable molecular structures whether under neutral or low pH environments. We also find that the salt bridges such as D177-R163 contribute to the structural stability of wild-type rabbit prion protein under neutral pH environment.

Conserved Stress-protective Activity between Prion Protein and Shadoo

Shadoo (Sho) is a neuronally expressed glycoprotein of unknown function. Although there is no overall sequence homology to the cellular prion protein (PrP(C)), both proteins contain a highly conserved internal hydrophobic domain (HD) and are tethered to the outer leaflet of the plasma membrane via a C-terminal glycosylphosphatidylinositol anchor. A previous study revealed that Sho can reduce toxicity of a PrP mutant devoid of the HD (PrPΔHD). We have now studied the stress-protective activity of Sho in detail and identified domains involved in this activity. Like PrP(C), Sho protects cells against physiological stressors such as the excitotoxin glutamate. Moreover, both PrP(C) and Sho required the N-terminal domain for this activity; the stress-protective capacity of PrPΔN as well as ShoΔN was significantly impaired. In both proteins, the HD promoted homodimer formation; however, deletion of the HD had different effects. Although ShoΔHD lost its stress-protective activity, PrPΔHD acquired a neurotoxic potential. Finally, we could show that the N-terminal domain of PrP(C) could be functionally replaced by that of Sho, suggesting a similar function of the N termini of Sho and PrP(C). Our study reveals a conserved physiological activity between PrP(C) and Sho to protect cells from stress-induced toxicity and suggests that Sho and PrP(C) might act on similar signaling pathways.

A Novel PrP Partner HS-1 Associated Protein X-1 (HAX-1) Protected the Cultured Cells Against the Challenge of H2O2

Prion protein (PrP) is a ubiquitous conserved glycoprotein predominantly expressed in neurons of the central nervous system (CNS). To elucidate on its cellular function, we performed a yeast two-hybrid screen within an adult human brain cDNA library for potential PrP-binding molecules. A novel protein, HS-1 associated protein X-1 (HAX-1), was identified to be able to bind with PrP strongly. The interaction between the two proteins has been further verified by glutathione-S-transferase (GST) pull-down and immunoprecipitation assays. The minimal binding regions were mapped to the segments of residues aa 91-163 for PrP(C) and residues aa 38-129 for HAX-1. Immunofluorescent assays of co-expressions of human PrP and HAX-1 in 293T and SHSY-5Y cells revealed marked co-localizations of those two proteins in cytoplasm. Moreover, the co-expression of HAX-1 and wild-type PrP (PG5) was found to enhance the cellular resistance to the challenge of H₂O₂. Contrarily, co-transfection of HAX-1 did not reverse but aggravated the cytotoxicities of the genetic CJD (gCJD) associated PrP mutants with nine- (PG9) and fourteen-octarepeats (PG14). Our data provide for the first time a new PrP-interacting partner that may play role in cell oxidative stress and anti-apoptosis physiologically and cell damage pathologically.

Emergence of multiple prion strains from single isolates of ovine scrapie

The infectious agent associated with prion diseases such as ovine scrapie shows strain diversity. Ovine prion strains have typically been identified by their transmission properties in wild-type mice. However, strain typing of ovine scrapie isolates in wild-type mice may not reveal properties of the infectious prion agent as they exist in the original host. This could be circumvented if ovine scrapie isolates are passaged in ovine prion protein (PrP)-transgenic mice. This study used incubation time, lesion profile, immunohistochemistry of the disease-associated PrP (PrP(Sc)) and molecular profile to compare the range of ovine prion strains that emerged from sheep scrapie isolates following serial passage in wild-type and ovine PrP transgenic mice. It was found that a diverse range of ovine prion strains emerged from homozygous ARQ and VRQ scrapie isolates passaged in wild-type and ovine PrP transgenic mice. However, strain-specific PrP(Sc) deposition and PrP27-30 molecular profile patterns were identified in ovine PrP transgenic mice that were not detected in wild-type mice. Significantly, it was established that the individual mouse brain selected for transmission during prion strain typing had a significant influence on strain definition. Serial passage of short- and long-incubation-time animals from the same group of scrapie-inoculated mice revealed different prion strain phenotypes. These observations are consistent with the possibility that some scrapie isolates contain more than one prion strain.

Prion Propagation in Cells Expressing PrP Glycosylation Mutants

Infection by prions involves conversion of a host-encoded cell surface protein (PrP(C)) to a disease-related isoform (PrP(Sc)). PrP(C) carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrP(C) glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrP(Sc) and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrP(Sc), while PrP(C) with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrP(C), were able to form infectious PrP(Sc). Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection.

A Drug-Based Cellular Assay (DBCA) for studying cytotoxic and cytoprotective activities of the prion protein: A practical guide

Although a great deal of progress has been made in elucidating the molecular identity of the infectious agent in prion diseases, the mechanisms by which prions kill neurons, and the role of the cellular prion protein (PrPC) in this process, remain enigmatic. A window into the normal function of PrPC, and how it can be corrupted to produce neurotoxic effects, is provided by a PrP deletion mutant called ΔCR, which produces a lethal phenotype when expressed in transgenic mice. In a previous study, we described the unusual observation that cells expressing ΔCR PrP are hyper-sensitive to the toxic effects of two cationic antibiotics (G418 and Zeocin) that are typically used for selection of transfected cell lines [1]. We have used this drug-sensitizing effect to develop a simple Drug-Based Cell Assay (DBCA) that reproduces several features of mutant PrP toxicity observed in vivo, including the rescuing activity of wild-type PrP. In this paper, we present a detailed guide for executing the DBCA in several, different experimental settings, including a new slot blot-based format. This assay provides a unique tool for studying PrP cytotoxic and cytoprotective activities in cell culture.

Compartment-restricted biotinylation reveals novel features of prion protein metabolism in vivo.

Proteins are often made in more than one form, with alternate versions sometimes residing in different cellular compartments than the primary species. The mammalian prion protein (PrP), a cell surface GPI-anchored protein, is a particularly noteworthy example for which minor cytosolic and transmembrane forms have been implicated in disease pathogenesis. To study these minor species, we used a selective labeling strategy in which spatially restricted expression of a biotinylating enzyme was combined with asymmetric engineering of the cognate acceptor sequence into PrP. Using this method, we could show that even wild-type PrP generates small amounts of the (Ctm)PrP transmembrane form. Selective detection of (Ctm)PrP allowed us to reveal its N-terminal processing, long half-life, residence in both intracellular and cell surface locations, and eventual degradation in the lysosome. Surprisingly, some human disease-causing mutants in PrP selectively stabilized (Ctm)PrP, revealing a previously unanticipated mechanism of (Ctm)PrP up-regulation that may contribute to disease. Thus, spatiotemporal tagging has uncovered novel aspects of normal and mutant PrP metabolism and should be readily applicable to the analysis of minor topologic isoforms of other proteins.

Pathogenic Mutations in the Hydrophobic Core of the Human Prion Protein Can Promote Structural Instability and Misfolding

Transmissible spongiform encephalopathies, or prion diseases, are caused by misfolding and aggregation of the prion protein PrP. These diseases can be hereditary in humans and four of the many disease-associated missense mutants of PrP are in the hydrophobic core: V180I, F198S, V203I and V210I. The T183A mutation is related to the hydrophobic core mutants as it is close to the hydrophobic core and known to cause instability. We used extensive molecular dynamics simulations of these five PrP mutants to compare their dynamics and conformations to those of the wild type PrP. The simulations highlight the changes that occur upon introduction of mutations and help to rationalize experimental findings. Changes can occur around the mutation site, but they can also be propagated over long distances. In particular, the F198S and T183A mutations lead to increased flexibility in parts of the structure that are normally stable, and the short β-sheet moves away from the rest of the protein. Mutations V180I, V210I and, to a lesser extent, V203I cause changes similar to those observed upon lowering the pH, which has been linked to misfolding. Early misfolding is observed in one V180I simulation. Overall, mutations in the hydrophobic core have a significant effect on the dynamics and stability of PrP, including the propensity to misfold, which helps to explain their role in the development of familial prion diseases.

Glycosaminoglycan Sulphation Affects the Seeded Misfolding of a Mutant Prion Protein

BackgroundThe accumulation of protease resistant conformers of the prion protein (PrPres) is a key pathological feature of prion diseases. Polyanions, including RNA and glycosaminoglycans have been identified as factors that contribute to the propagation, transmission and pathogenesis of prion disease. Recent studies have suggested that the contribution of these cofactors to prion propagation may be species specific.Methodology/Principal FindingIn this study a cell-free assay was used to investigate the molecular basis of polyanion stimulated PrPres formation using brain tissue or cell line derived murine PrP. Enzymatic depletion of endogenous nucleic acids or heparan sulphate (HS) from the PrPC substrate was found to specifically prevent PrPres formation seeded by mouse derived PrPSc. Modification of the negative charge afforded by the sulphation of glycosaminoglycans increased the ability of a familial PrP mutant to act as a substrate for PrPres formation, while having no effect on PrPres formed by wildtype PrP. This difference may be due to the observed differences in the binding of wild type and mutant PrP for glycosaminoglycans.Conclusions/SignificanceCofactor requirements for PrPres formation are host species and prion strain specific and affected by disease associated mutations of the prion protein. This may explain both species and strain dependent propagation characteristics and provide insights into the underlying mechanisms of familial prion disease. It further highlights the challenge of designing effective therapeutics against a disease which effects a range of mammalian species, caused by range of aetiologies and prion strains.

Neurotoxic Mutants of the Prion Protein Induce Spontaneous Ionic Currents in Cultured Cells

The mechanisms by which prions kill neurons and the role of the cellular prion protein in this process are enigmatic. Insight into these questions is provided by the neurodegenerative phenotypes of transgenic mice expressing prion protein (PrP) molecules with deletions of conserved amino acids in the central region. We report here that expression in transfected cells of the most toxic of these PrP deletion mutants (Delta105-125) induces large, spontaneous ionic currents that can be detected by patch-clamping techniques. These currents are produced by relatively non-selective, cation-permeable channels or pores in the cell membrane and can be silenced by overexpression of wild-type PrP, as well as by treatment with a sulfated glycosaminoglycan. Similar currents are induced by PrP molecules carrying several different point mutations in the central region that cause familial prion diseases in humans. The ionic currents described here are distinct from those produced in artificial lipid membranes by synthetic peptides derived from the PrP sequence because they are induced by membrane-anchored forms of PrP that are synthesized by cells and that are found in vivo. Our results indicate that the neurotoxicity of some mutant forms of PrP is attributable to enhanced ion channel activity and that wild-type PrP possesses a channel-silencing activity. Drugs that block PrP-associated channels or pores may therefore represent novel therapeutic agents for treatment of patients with prion diseases.

NMR Structure of the Human Prion Protein with the Pathological Q212P Mutation Reveals Unique Structural Features

Prion diseases are fatal neurodegenerative disorders caused by an aberrant accumulation of the misfolded cellular prion protein (PrPC) conformer, denoted as infectious scrapie isoform or PrPSc. In inherited human prion diseases, mutations in the open reading frame of the PrP gene (PRNP) are hypothesized to favor spontaneous generation of PrPSc in specific brain regions leading to neuronal cell degeneration and death. Here, we describe the NMR solution structure of the truncated recombinant human PrP from residue 90 to 231 carrying the Q212P mutation, which is believed to cause Gerstmann-Sträussler-Scheinker (GSS) syndrome, a familial prion disease. The secondary structure of the Q212P mutant consists of a flexible disordered tail (residues 90–124) and a globular domain (residues 125–231). The substitution of a glutamine by a proline at the position 212 introduces novel structural differences in comparison to the known wild-type PrP structures. The most remarkable differences involve the C-terminal end of the protein and the β2–α2 loop region. This structure might provide new insights into the early events of conformational transition of PrPC into PrPSc. Indeed, the spontaneous formation of prions in familial cases might be due to the disruptions of the hydrophobic core consisting of β2–α2 loop and α3 helix.

A Highly Toxic Cellular Prion Protein Induces a Novel, Nonapoptotic Form of Neuronal Death

Several different deletions within the N-terminal tail of the prion protein (PrP) induce massive neuronal death when expressed in transgenic mice. This toxicity is dose-dependently suppressed by coexpression of full-length PrP, suggesting that it results from subversion of a normal physiological activity of cellular PrP. We performed a combined biochemical and morphological analysis of Tg(DeltaCR) mice, which express PrP carrying a 21-aa deletion (residues 105-125) within a highly conserved region of the protein. Death of cerebellar granule neurons in Tg(DeltaCR) mice is not accompanied by activation of either caspase-3 or caspase-8 or by increased levels of the autophagy marker, LC3-II. In electron micrographs, degenerating granule neurons displayed a unique morphology characterized by heterogeneous condensation of the nuclear matrix without formation of discrete chromatin masses typical of neuronal apoptosis. Our data demonstrate that perturbations in PrP functional activity induce a novel, nonapoptotic, nonautophagic form of neuronal death whose morphological features are reminiscent of those associated with excitotoxic stress.

Changing the solvent accessibility of the prion protein disulfide bond markedly influences its trafficking and effect on cell function

Prion diseases are fatal transmissible neurodegenerative diseases that result from structural conversion of the prion protein into a disease-associated isoform. The prion protein contains a single disulfide bond. Our analysis of all NMR structures of the prion protein (total of 440 structures over nine species) containing an explicit disulfide bond reveals that the bond exists predominantly in a stable low-energy state, but can also adopt a high-energy configuration. The side chains of two tyrosine residues and one phenylalanine residue control access of solvent to the disulfide bond. Notably, the side chains rotate away from the disulfide bond in the high-energy state, exposing the disulfide bond to solvent. The importance of these aromatic residues for protein function was analysed by mutating them to alanine residues and analysing the properties of the mutant proteins using biophysical and cell biological approaches. Whereas the mutant protein behaved similarly to wild-type prion protein in recombinant systems, the mutants were retained in the endoplasmic reticulum of mammalian cells and degraded by the proteasomal system. The cellular behaviour of the aromatic residue mutants was similar to the cellular behaviour of a disulfide bond mutant prion protein in which the cysteine residues were replaced with alanine, a result which is consistent with an unstable disulfide bond in the aromatic residue mutants. These observations suggest that the conformation of the prion protein disulfide bond may have implications for correct maturation and function of this protein.

Neuron Dysfunction Is Induced by Prion Protein with an Insertional Mutation via a Fyn Kinase and Reversed by Sirtuin Activation inCaenorhabditis elegans

Although prion propagation is well understood, the signaling pathways activated by neurotoxic forms of prion protein (PrP) and those able to mitigate pathological phenotypes remain largely unknown. Here, we identify src-2, a Fyn-related kinase, as a gene required for human PrP with an insertional mutation to be neurotoxic in Caenorhabditis elegans, and the longevity modulator sir-2.1/SIRT1, a sirtuin deacetylase, as a modifier of prion neurotoxicity. The expression of octarepeat-expanded PrP in C. elegans mechanosensory neurons led to a progressive loss of response to touch without causing cell death, whereas wild-type PrP expression did not alter behavior. Transgenic PrP molecules showed expression at the plasma membrane, with protein clusters, partial resistance to proteinase K (PK), and protein insolubility detected for mutant PrP. Loss of function (LOF) of src-2 greatly reduced mutant PrP neurotoxicity without reducing PK-resistant PrP levels. Increased sir-2.1 dosage reversed mutant PrP neurotoxicity, whereas sir-2.1 LOF showed aggravation, and these effects did not alter PK-resistant PrP. Resveratrol, a polyphenol known to act through sirtuins for neuroprotection, reversed mutant PrP neurotoxicity in a sir-2.1-dependent manner. Additionally, resveratrol reversed cell death caused by mutant PrP in cerebellar granule neurons from prnp-null mice. These results suggest that Fyn mediates mutant PrP neurotoxicity in addition to its role in cellular PrP signaling and reveal that sirtuin activation mitigates these neurotoxic effects. Sirtuin activators may thus have therapeutic potential to protect from prion neurotoxicity and its effects on intracellular signaling.