Familial Prion Diseases Research Articles

Familial prion protein mutants inhibit Hrd1-mediated retrotranslocation of misfolded proteins by depleting misfolded protein sensor BiP.

Similar to many proteins trafficking through the secretory pathway, cellular prion protein (PrP) partly retrotranslocates from the endoplasmic reticulum to the cytosol through the endoplasmic reticulum-associated degradation (ERAD) pathway in an attempt to alleviate accumulation of cellular misfolded PrP. Surprisingly, familial PrP mutants fail to retrotranslocate and simultaneously block normal cellular PrP retrotranslocation. That impairments in retrotranslocation of misfolded proteins could lead to global disruptions in cellular homeostasis prompted further investigations into PrP mutant retrotranslocation defects. A gain- and loss-of-function approach identified human E3 ubiquitin ligase, Hrd1, as a critical regulator of PrP retrotranslocation in mammalian cells. Expression of familial human PrP mutants, V210I(129V) and M232R(129V), not only abolished PrP retrotranslocation, but also that of Hrd1-dependent ERAD substrates, transthyretin TTR(D18G) and α1-anti-trypsin A1AT(NHK). Mutant PrP expression decreased binding immunoglobulin protein (BiP) levels by 50% and attenuated ER stress-induced BiP by increasing BiP turnover 6-fold. Overexpression of BiP with PrP mutants rescued retrotranslocation of PrP, TTR(D18G) and A1AT(NHK). PrP mutants-induced cell death was also rescued by co-expression of BiP. These results show that PrP mutants highjack the Hrd1-dependent ERAD pathway, an action that would result in misfolded protein accumulation especially in terminally differentiated neurons. This could explain the age-dependent neuronal degeneration in familial prion diseases.

A Case of Familial Prion Disease Diagnosed as Alzheimer’s Disease for 5 Years

A Case of Familial Prion Disease Diagnosed as Alzheimer’s Disease for 5 Years

Characterization of the first American case of familial prion disease due to the PRNP-G114V mutation (P4.103)

Characterization of the first American case of familial prion disease due to the PRNP-G114V mutation (P4.103)

β-hairpin-mediated formation of structurally distinct multimers of neurotoxic prion peptides.

Protein misfolding disorders are associated with conformational changes in specific proteins, leading to the formation of potentially neurotoxic amyloid fibrils. During pathogenesis of prion disease, the prion protein misfolds into β-sheet rich, protease-resistant isoforms. A key, hydrophobic domain within the prion protein, comprising residues 109–122, recapitulates many properties of the full protein, such as helix-to-sheet structural transition, formation of fibrils and cytotoxicity of the misfolded isoform. Using all-atom, molecular simulations, it is demonstrated that the monomeric 109–122 peptide has a preference for α-helical conformations, but that this peptide can also form β-hairpin structures resulting from turns around specific glycine residues of the peptide. Altering a single amino acid within the 109–122 peptide (A117V, associated with familial prion disease) increases the prevalence of β-hairpin formation and these observations are replicated in a longer peptide, comprising residues 106–126. Multi-molecule simulations of aggregation yield different assemblies of peptide molecules composed of conformationally-distinct monomer units. Small molecular assemblies, consistent with oligomers, comprise peptide monomers in a β-hairpin-like conformation and in many simulations appear to exist only transiently. Conversely, larger assemblies are comprised of extended peptides in predominately antiparallel β-sheets and are stable relative to the length of the simulations. These larger assemblies are consistent with amyloid fibrils, show cross-β structure and can form through elongation of monomer units within pre-existing oligomers. In some simulations, assemblies containing both β-hairpin and linear peptides are evident. Thus, in this work oligomers are on pathway to fibril formation and a preference for β-hairpin structure should enhance oligomer formation whilst inhibiting maturation into fibrils. These simulations provide an important new atomic-level model for the formation of oligomers and fibrils of the prion protein and suggest that stabilization of β-hairpin structure may enhance cellular toxicity by altering the balance between oligomeric and fibrillar protein assemblies.

Distinct patterns of spread of prion infection in brains of mice expressing anchorless or anchored forms of prion protein

BackgroundIn humans and animals, prion protein (PrP) is usually expressed as a glycophosphatidylinositol (GPI)-anchored membrane protein, but anchorless PrP may be pathogenic in humans with certain familial prion diseases. Anchored PrP expressed on neurons mediates spread of prions along axons in the peripheral and central nervous systems. However, the mechanism of prion spread in individuals expressing anchorless PrP is poorly understood. Here we studied prion spread within brain of mice expressing anchorless or anchored PrP.ResultsTo create a localized initial point of infection, we microinjected scrapie in a 0.5 microliter volume in the striatum. In this experiment, PrPres and gliosis were first detected in both types of mice at 40 days post-inoculation near the needle track. In mice with anchored PrP, PrPres appeared to spread via neurons to distant connected brain areas by the clinical endpoint at 150 days post-inoculation. This PrPres was rarely associated with blood vessels. In contrast, in mice with anchorless PrP, PrPres spread did not follow neuronal circuitry, but instead followed a novel slower pattern utilizing the drainage system of the brain interstitial fluid (ISF) including perivascular areas adjacent to blood vessels, subependymal areas and spaces between axons in white matter tracts.ConclusionsIn transgenic mice expressing anchorless PrP small amyloid-seeding PrPres aggregates appeared to be transported in the ISF, thus spreading development of cerebral amyloid angiopathy (CAA) throughout the brain. Spread of amyloid seeding by ISF may also occur in multiple human brain diseases involving CAA.

Genetic studies in human prion diseases.

Human prion diseases are fatal neurodegenerative disorders that are characterized by spongiform changes, astrogliosis, and the accumulation of an abnormal prion protein (PrP(Sc)). Approximately 10%-15% of human prion diseases are familial variants that are caused by pathogenic mutations in the prion protein gene (PRNP). Point mutations or the insertions of one or more copies of a 24 bp repeat are associated with familial human prion diseases including familial Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker syndrome, and fatal familial insomnia. These mutations vary significantly in frequency between countries. Here, we compare the frequency of PRNP mutations between European countries and East Asians. Associations between single nucleotide polymorphisms (SNPs) of several candidate genes including PRNP and CJD have been reported. The SNP of PRNP at codon 129 has been shown to be associated with sporadic, iatrogenic, and variant CJD. The SNPs of several genes other than PRNP have been showed contradictory results. Case-control studies and genome-wide association studies have also been performed to identify candidate genes correlated with variant and/or sporadic CJD. This review provides a general overview of the genetic mutations and polymorphisms that have been analyzed in association with human prion diseases to date.

De Novo Prion Aggregates Trigger Autophagy in Skeletal Muscle

In certain sporadic, familial, and infectious prion diseases, the prion protein misfolds and aggregates in skeletal muscle in addition to the brain and spinal cord. In myocytes, prion aggregates accumulate intracellularly, yet little is known about clearance pathways. Here we investigated the clearance of prion aggregates in muscle of transgenic mice that develop prion disease de novo. In addition to neurodegeneration, aged mice developed a degenerative myopathy, with scattered myocytes containing ubiquitinated, intracellular prion inclusions that were adjacent to myocytes lacking inclusions. Myocytes also showed elevated levels of the endoplasmic reticulum chaperone Grp78/BiP, suggestive of impaired protein degradation and endoplasmic reticulum stress. Additionally, autophagy was induced, as indicated by increased levels of beclin-1 and LC3-II. In C2C12 myoblasts, inhibition of autophagosome maturation or lysosomal degradation led to enhanced prion aggregation, consistent with a role for autophagy in prion aggregate clearance. Taken together, these findings suggest that the induction of autophagy may be a central strategy for prion aggregate clearance in myocytes. IMPORTANCE In prion diseases, the prion protein misfolds and aggregates in the central nervous system and sometimes in other organs, including muscle, yet the cellular pathways of prion aggregate clearance are unclear. Here we investigated the clearance of prion aggregates in the muscle of a transgenic mouse model that develops profound muscle degeneration. We found that endoplasmic reticulum stress pathways were activated and that autophagy was induced. Blocking of autophagic degradation in cell culture models led to an accumulation of aggregated prion protein. Collectively, these findings suggest that autophagy has an instrumental role in prion protein clearance.

Association of prion protein genotype and scrapie prion protein type with cellular prion protein charge isoform profiles in cerebrospinal fluid of humans with sporadic or familial prion diseases

The present study investigates whether posttranslational modifications of cellular prion protein (PrPC) in the cerebrospinal fluid (CSF) of humans with prion diseases are associated with methionine (M) and/or valine (V) polymorphism at codon 129 of the prion protein gene (PRNP), scrapie prion protein (PrPSc) type in sporadic Creutzfeldt-Jakob disease (sCJD), or PRNP mutations in familial Creutzfeldt-Jakob disease (fCJD/E200K), and fatal familial insomnia (FFI). We performed comparative 2-dimensional immunoblotting of PrPC charge isoforms in CSF samples from cohorts of diseased and control donors. Mean levels of total PrPC were significantly lower in the CSF from fCJD patients than from those with sCJD or FFI. Of the 12 most abundant PrPC isoforms in the examined CSF, one (IF12) was relatively decreased in (1) sCJD with VV (vs. MM or MV) at PRNP codon 129; (2) in sCJD with PrPSc type 2 (vs. PrPSc type 1); and (3) in FFI versus sCJD or fCJD. Furthermore, truncated PrPC species were detected in sCJD and control samples without discernible differences. Finally, serine 43 of PrPC in the CSF and brain tissue from CJD patients showed more pronounced phosphorylation than in control donors.

A novel familial prion disease causing pan‐autonomic‐sensory neuropathy and cognitive impairment

A novel familial prion disease causing pan‐autonomic‐sensory neuropathy and cognitive impairment

Unique drug screening approach for prion diseases identifies tacrolimus and astemizole as antiprion agents

Prion diseases such as Creutzfeldt-Jakob disease (CJD) are incurable and rapidly fatal neurodegenerative diseases. Because prion protein (PrP) is necessary for prion replication but dispensable for the host, we developed the PrP-FRET-enabled high throughput assay (PrP-FEHTA) to screen for compounds that decrease PrP expression. We screened a collection of drugs approved for human use and identified astemizole and tacrolimus, which reduced cell-surface PrP and inhibited prion replication in neuroblastoma cells. Tacrolimus reduced total cellular PrP levels by a nontranscriptional mechanism. Astemizole stimulated autophagy, a hitherto unreported mode of action for this pharmacophore. Astemizole, but not tacrolimus, prolonged the survival time of prion-infected mice. Astemizole is used in humans to treat seasonal allergic rhinitis in a chronic setting. Given the absence of any treatment option for CJD patients and the favorable drug characteristics of astemizole, including its ability to cross the blood-brain barrier, it may be considered as therapy for CJD patients and for prophylactic use in familial prion diseases. Importantly, our results validate PrP-FEHTA as a method to identify antiprion compounds and, more generally, FEHTA as a unique drug discovery platform.

Non‐amyloid and amyloid prion protein deposits in prion‐infected mice differ in blockage of interstitial brain fluid

Prion diseases are characterized by brain deposits of misfolded aggregated protease-resistant prion protein (PrP), termed PrPres. In humans and animals, PrPres is found as either disorganized non-amyloid aggregates or organized amyloid fibrils. Both PrPres forms are found in extracellular spaces of the brain. Thus, both might block drainage of brain interstitial fluid (ISF). The present experiments studied whether ISF blockage occurred during amyloid and/or non-amyloid prion diseases. Various-sized fluorescein-labelled ISF tracers were stereotactically inoculated into the striatum of adult mice. At times from 5 min to 77 h, uninfected and scrapie-infected mice were compared. C57BL/10 mice expressing wild-type anchored PrP, which develop non-amyloid PrPres similar to humans with sporadic Creutzfeldt-Jakob disease, were compared with Tg44+/+ mice (transgenic mice secreting anchorless PrP) expressing anchorless PrP, which develop amyloid PrPres similar to certain human familial prion diseases. In C57BL/10 mice, extensive non-amyloid PrPres aggregate deposition was not associated with abnormal clearance kinetics of tracers. In contrast, scrapie-infected Tg44+/+ mice showed blockage of tracer clearance and colocalization of tracer with perivascular PrPres amyloid. As tracer localization and clearance was normal in infected C57BL/10 mice, ISF blockage was not an important pathogenic mechanism in this model. Therefore, ISF blockage is unlikely to be a problem in non-amyloid human prion diseases such as sporadic Creutzfeldt-Jakob disease. In contrast, partial ISF blockage appeared to be a possible pathogenic mechanism in Tg44+/+ mice. Thus this mechanism might also influence human amyloid prion diseases where expression of anchorless or mutated PrP results in perivascular amyloid PrPres deposition and cerebral amyloid angiopathy.

Burial of the Polymorphic Residue 129 in Amyloid Fibrils of Prion Stop Mutants

Misfolding of the natively α-helical prion protein into a β-sheet rich isoform is related to various human diseases such as Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker syndrome. In humans, the disease phenotype is modified by a methionine/valine polymorphism at codon 129 of the prion protein gene. Using a combination of hydrogen/deuterium exchange coupled to NMR spectroscopy, hydroxyl radical probing detected by mass spectrometry, and site-directed mutagenesis, we demonstrate that stop mutants of the human prion protein have a conserved amyloid core. The 129 residue is deeply buried in the amyloid core structure, and its mutation strongly impacts aggregation. Taken together the data support a critical role of the polymorphic residue 129 of the human prion protein in aggregation and disease.

Zinc Drives a Tertiary Fold in the Prion Protein with Familial Disease Mutation Sites at the Interface

The cellular prion protein PrP(C) consists of two domains--a flexible N-terminal domain, which participates in copper and zinc regulation, and a largely helical C-terminal domain that converts to β sheet in the course of prion disease. These two domains are thought to be fully independent and noninteracting. Compelling cellular and biophysical studies, however, suggest a higher order structure that is relevant to both PrP(C) function and misfolding in disease. Here, we identify a Zn²⁺-driven N-terminal to C-terminal tertiary interaction in PrP(C). The C-terminal surface participating in this interaction carries the majority of the point mutations that confer familial prion disease. Investigation of mutant PrPs finds a systematic relationship between the type of mutation and the apparent strength of this domain structure. The structural features identified here suggest mechanisms by which physiologic metal ions trigger PrP(C) trafficking and control prion disease.

Molecular Genetics of Gerstmann-Straussler-Scheinker Disease and Creutzfeld-Jakob Disease

Prion diseases are a group of transmissible neurodegenerative disorders associated with the misfolded form of the prion protein, PrPSc . The latter isoform is derived by conformational conversion of the normal prion protein, PrPc . The gene encoding the prion protein is highly conserved among species. There are several distinct types of prion diseases in humans: kuru, Creutzfeldt - Jakob disease (CJD), Gerstmann-Straussler-Scheinker disease (GSS) and Fatal Familial Insomnia (FFI) and its sporadic analogue, fatal sporadic insomnia. While human prion diseases are mostly sporadic, some 15% of CJD and all cases of GSS are hereditary disorders caused by mutations in the PRNP gene. There are two major types of mutations in PRNP : point mutations leading to amino acid substitutions and mutations leading to expansions of the octapeptide repeat region within the N-terminal part of the prion protein. This review describes different types of familial prion diseases linked to specific polymorphisms and mutation in PRNP

Failure of Prion Protein Oxidative Folding Guides the Formation of Toxic Transmembrane Forms

The mechanism by which pathogenic mutations in the globular domain of the cellular prion protein (PrP(C)) increase the likelihood of misfolding and predispose to diseases is not yet known. Differences in the evidences provided by structural and metabolic studies of these mutants suggest that in vivo folding could be playing an essential role in their pathogenesis. To address this role, here we use the single or combined M206S and M213S artificial mutants causing labile folds and express them in cells. We find that these mutants are highly toxic, fold as transmembrane PrP, and lack the intramolecular disulfide bond. When the mutations are placed in a chain with impeded transmembrane PrP formation, toxicity is rescued. These results suggest that oxidative folding impairment, as on aging, can be fundamental for the genesis of intracellular neurotoxic intermediates key in prion neurodegenerations.

D02 A case of Huntington disease-like 1 (HDL1) with long disease duration

<h3>Summary</h3> Dynamic control of cell polarity is of critical importance for many aspects of cellular development and motility. In <i>Myxococcus xanthus</i>, a G-protein and its cognate GTPase-activating protein establish a polarity axis that defines the direction of movement of the cell and which can be rapidly inverted by the Frz chemosensory system. Although vital for collective cell behaviours, how Frz triggers this switch has remained unknown. Here, we use genetics, imaging and mathematical modelling to show that Frz controls polarity reversals via a gated relaxation oscillator. FrzX, which we newly identify as the primary Frz output, provides the gating and thus acts as the trigger for reversals. Slow relocalisation of the polarity protein RomR then creates a refractory period during which another switch cannot be triggered. A secondary Frz output, FrzZ, decreases this delay allowing rapid reversals when required. This architecture thus results in a highly tunable switch that allows a wide range of motility responses.

O4‐05‐04: A novel familial prion disease causing cognitive impairment, polyneuropathy and pan‐autonomic involvement

O4‐05‐04: A novel familial prion disease causing cognitive impairment, polyneuropathy and pan‐autonomic involvement

Exploring structural and thermodynamic stabilities of human prion protein pathogenic mutants D202N, E211Q and Q217R

The central event in the pathogenesis of prion protein (PrP) is a profound conformational change from its α-helical (PrP(C)) to its β-sheet-rich isoform (PrP(Sc)). Many single amino acid mutations of PrP are associated with familial prion diseases, such as D202N, E211Q, and Q217R mutations located at the third native α-helix of human PrP. In order to explore the underlying structural and dynamic effects of these mutations, we performed all-atom molecular dynamics (MD) simulations for the wild-type (WT) PrP and its mutants. The obtained results indicate that these amino acid substitutions have subtle effects on the protein structures, but show large changes of the overall electrostatic potential distributions. We can infer that the changes of PrP electrostatic surface due to the studied mutations may influence the intermolecular interactions during the aggregation process. In addition, the mutations also affect the thermodynamic stabilities of PrP.

An overview of human prion diseases

Prion diseases are transmissible, progressive and invariably fatal neurodegenerative conditions associated with misfolding and aggregation of a host-encoded cellular prion protein, PrPC. They have occurred in a wide range of mammalian species including human. Human prion diseases can arise sporadically, be hereditary or be acquired. Sporadic human prion diseases include Cruetzfeldt-Jacob disease (CJD), fatal insomnia and variably protease-sensitive prionopathy. Genetic or familial prion diseases are caused by autosomal dominantly inherited mutations in the gene encoding for PrPC and include familial or genetic CJD, fatal familial insomnia and Gerstmann-Sträussler-Scheinker syndrome. Acquired human prion diseases account for only 5% of cases of human prion disease. They include kuru, iatrogenic CJD and a new variant form of CJD that was transmitted to humans from affected cattle via meat consumption especially brain. This review presents information on the epidemiology, etiology, clinical assessment, neuropathology and public health concerns of human prion diseases. The role of the PrP encoding gene (PRNP) in conferring susceptibility to human prion diseases is also discussed.

Structural polymorphism in amyloids: new insights from studies with Y145Stop prion protein fibrils.

The C-terminally-truncated human prion protein variant Y145Stop (or PrP23-144), associated with a familial prion disease, provides a valuable model for studying the fundamental properties of protein amyloids. In previous solid-state NMR experiments, we established that the β-sheet core of the PrP23-144 amyloid is composed of two β-strand regions encompassing residues ∼113-125 and ∼130-140. The former segment contains a highly conserved hydrophobic palindrome sequence, (113)AGAAAAGA(120), which has been considered essential to PrP conformational conversion. Here, we examine the role of this segment in fibrillization of PrP23-144 using a deletion variant, Δ113-120 PrP23-144, in which the palindrome sequence is missing. Surprisingly, we find that deletion of the palindrome sequence affects neither the amyloidogenicity nor the polymerization kinetics of PrP23-144, although it does alter amyloid conformation and morphology. Using two-dimensional and three-dimensional solid-state NMR methods, we find that Δ113-120 PrP23-144 fibrils contain an altered β-core extended N-terminally to residue ∼106, encompassing residues not present in the core of wild-type PrP23-144 fibrils. The C-terminal β-strand of the core, however, is similar in both fibril types. Collectively, these data indicate that amyloid cores of PrP23-144 variants contain "essential" (i.e. nucleation-determining) and "nonessential" regions, with the latter being "movable" in amino acid sequence space. These findings reveal an intriguing new mechanism for structural polymorphism in amyloids and suggest a potential means for modulating the physicochemical properties of amyloid fibrils without compromising their polymerization characteristics.