Amyloid Core Formed of Full-Length Recombinant Mouse Prion Protein Involves Sequence 127–143 but Not Sequence 107–126

The principal event underlying the development of prion disease is the conversion of soluble cellular prion protein (PrP^C) into its disease-causing isoform, PrP^Sc. This conversion is associated with a marked change in secondary structure from predominantly α-helical to a high β-sheet content, ultimately leading to the formation of aggregates consisting of ordered fibrillar assemblies referred to as amyloid. In vitro, recombinant prion proteins and short prion peptides from various species have been shown to form amyloid under various conditions and it has been proposed that, theoretically, any protein and peptide could form amyloid under appropriate conditions. To identify the peptide segment involved in the amyloid core formed from recombinant full-length mouse prion protein mPrP(23–230), we carried out seed-induced amyloid formation from recombinant prion protein in the presence of seeds generated from the short prion peptides mPrP(107–143), mPrP(107–126), and mPrP(127–143). Our results showed that the amyloid fibrils formed from mPrP(107–143) and mPrP(127–143), but not those formed from mPrP(107–126), were able to seed the amyloidogenesis of mPrP(23–230), showing that the segment residing in sequence 127–143 was used to form the amyloid core in the fibrillization of mPrP(23–230).     

Conformational Stability of Mammalian Prion Protein Amyloid Fibrils Is Dictated by a Packing Polymorphism within the Core Region

Mammalian prion strains are believed to arise from the propagation of distinct conformations of the misfolded prion protein PrP^Sc. One key operational parameter used to define differences between strains has been conformational stability of PrP^Sc as defined by resistance to thermal and/or chemical denaturation. However, the structural basis of these stability differences is unknown. To bridge this gap, we have generated two strains of recombinant human prion protein amyloid fibrils that show dramatic differences in conformational stability and have characterized them by a number of biophysical methods. Backbone amide hydrogen/deuterium exchange experiments revealed that, in sharp contrast to previously studied strains of infectious amyloid formed from the yeast prion protein Sup35, differences in β-sheet core size do not underlie differences in conformational stability between strains of mammalian prion protein amyloid. Instead, these stability differences appear to be dictated by distinct packing arrangements (i.e. steric zipper interfaces) within the amyloid core, as indicated by distinct x-ray fiber diffraction patterns and large strain-dependent differences in hydrogen/deuterium exchange kinetics for histidine side chains within the core region. Although this study was limited to synthetic prion protein amyloid fibrils, a similar structural basis for strain-dependent conformational stability may apply to brain-derived PrP^Sc, especially because large strain-specific differences in PrP^Sc stability are often observed despite a similar size of the PrP^Sc core region.     

Crystallographic Studies of Prion Protein (PrP) Segments Suggest How Structural Changes Encoded by Polymorphism at Residue 129 Modulate Susceptibility to Human Prion Disease

A single nucleotide polymorphism (SNP) in codon 129 of the human prion gene, leading to a change from methionine to valine at residue 129 of prion protein (PrP), has been shown to be a determinant in the susceptibility to prion disease. However, the molecular basis of this effect remains unexplained. In the current study, we determined crystal structures of prion segments having either Met or Val at residue 129. These 6-residue segments of PrP centered on residue 129 are “steric zippers,” pairs of interacting β-sheets. Both structures of these “homozygous steric zippers” reveal direct intermolecular interactions between Met or Val in one sheet and the identical residue in the mating sheet. These two structures, plus a structure-based model of the heterozygous Met-Val steric zipper, suggest an explanation for the previously observed effects of this locus on prion disease susceptibility and progression.     

Unaltered prion protein expression in Alzheimer disease patients

The suggested role of cellular prion protein (PrP^C) in mediating the toxic effects of oligomeric amyloid β peptide (Aβ) in Alzheimer disease (AD) is controversial. To address the hypothesis that variable PrP^C expression is involved in AD pathogenesis, we analyzed PrP^C expression in the frontal and temporal cortices and hippocampus of individuals with no cognitive impairment (NCI), amnestic mild cognitive impairment (aMCI), mild AD (mAD) and AD. We found that PrP^C expression in all brain regions was not significantly altered among the various patient groups. In addition, PrP^C levels in all groups did not correlate with expression of methionine (M) or valine (V) at codon 129 of the PrP gene, a polymorphism that has been linked in some studies to increased risk for AD, and which occurs in close proximity to the proposed binding region for the oligomeric Aβ peptide. Our results indicate that, if PrP^C is involved in mediating the toxic effects of the oligomeric Aβ peptide, these effects occur independently of steady state levels of PrP or the codon 129 polymorphism.Key words: prion protein, PrP codon 129 polymorphism, Alzheimer disease, oligomeric Aβ, Alzheimer precursor protein     

Prion infection promotes extensive accumulation of α-synuclein in aged human α-synuclein transgenic mice

In neurodegenerative disorders of the aging population, misfolded proteins, such as PrP^Sc, α-synuclein, amyloid β protein and tau, can interact resulting in enhanced aggregation, cross seeding and accelerated disease progression. Previous reports have shown that in Creutzfeldt-Jakob disease and scrapie, α-synuclein accumulates near PrP^Sc deposits. However, it is unclear if pre-existing human α-synuclein aggregates modified prion disease pathogenesis, or if PrP^Sc exacerbates the α-synuclein pathology. Here, we inoculated infectious prions into aged α-synuclein transgenic (tg) and non-transgenic littermate control mice by the intracerebral route. Remarkably, inoculation of RML and mNS prions into α-synuclein tg mice resulted in more extensive and abundant intraneuronal and synaptic α-synuclein accumulation. In addition, infectious prions led to the formation of perineuronal α-synuclein deposits with a neuritic plaque-like appearance. Prion pathology was unmodified by the presence of α-synuclein. However, with the mNS prion strain there was a modest but significant acceleration in the time to terminal prion disease in mice having α-synuclein aggregates as compared with non-tg mice. Taken together, these studies support the notion that PrP^Sc directly or indirectly promotes α-synuclein pathology.     

Left handed �� helix models for mammalian prion fibrils

We propose models for in vitro grown mammalian prion protein fibrils based upon left handed beta helices formed both from the N-terminal and C-terminal regions of the proteinase resistant infectious prion core. The C-terminal threading onto a β-helical structure is almost uniquely determined by fixing the cysteine disulfide bond on a helix corner. In comparison to known left handed helical peptides, the resulting model structures have similar stability attributes including relatively low root mean square deviations in all atom molecular dynamics, substantial side-chain-to-side-chain hydrogen bonding, good volume packing fraction, and low hydrophilic/hydrophobic frustration. For the N-terminus, we propose a new threading of slightly more than two turns, which improves upon the above characteristics relative to existing three turn β-helical models. The N-terminal and C-terminal beta helices can be assembled into eight candidate models for the fibril repeat units, held together by large hinge (order 30 residues) domain swapping, with three amenable to fibril promoting domain swapping via a small (five residue) hinge on the N-terminal side. Small concentrations of the metastable C-terminal β helix in vivo might play a significant role in templating the infectious conformation and in enhancing conversion kinetics for inherited forms of the disease and explain resistance (for canines) involving hypothesized coupling to the methionine 129 sulfur known to play a role in human disease.Key words: prion, amyloid fibril, domain swap, beta helix, computational biology     

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.     

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 (PrP^C) conformer, denoted as infectious scrapie isoform or PrP^Sc. In inherited human prion diseases, mutations in the open reading frame of the PrP gene (PRNP) are hypothesized to favor spontaneous generation of PrP^Sc 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 PrP^C into PrP^Sc. 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.     

Prion Domain of Yeast Ure2 Protein Adopts a Completely Disordered Structure: A Solid-Support EPR Study

Amyloid fibril formation is associated with a range of neurodegenerative diseases in humans, including Alzheimer’s, Parkinson’s, and prion diseases. In yeast, amyloid underlies several non-Mendelian phenotypes referred to as yeast prions. Mechanism of amyloid formation is critical for a complete understanding of the yeast prion phenomenon and human amyloid-related diseases. Ure2 protein is the basis of yeast prion [URE3]. The Ure2p prion domain is largely disordered. Residual structures, if any, in the disordered region may play an important role in the aggregation process. Studies of Ure2p prion domain are complicated by its high aggregation propensity, which results in a mixture of monomer and aggregates in solution. Previously we have developed a solid-support electron paramagnetic resonance (EPR) approach to address this problem and have identified a structured state for the Alzheimer’s amyloid-β monomer. Here we use solid-support EPR to study the structure of Ure2p prion domain. EPR spectra of Ure2p prion domain with spin labels at every fifth residue from position 10 to position 75 show similar residue mobility profile for denaturing and native buffers after accounting for the effect of solution viscosity. These results suggest that Ure2p prion domain adopts a completely disordered structure in the native buffer. A completely disordered Ure2p prion domain implies that the amyloid formation of Ure2p, and likely other Q/N-rich yeast prion proteins, is primarily driven by inter-molecular interactions.     

Soluble Prion Protein Inhibits Amyloid-β (Aβ) Fibrillization and Toxicity

The pathogenesis of Alzheimer disease appears to be strongly linked to the aggregation of amyloid-β (Aβ) peptide and, especially, formation of soluble Aβ1–42 oligomers. It was recently demonstrated that the cellular prion protein, PrP^C, binds with high affinity to these oligomers, acting as a putative receptor that mediates at least some of their neurotoxic effects. Here we show that the soluble (i.e. glycophosphatidylinositol anchor-free) prion protein and its N-terminal fragment have a strong effect on the aggregation pathway of Aβ1–42, inhibiting its assembly into amyloid fibrils. Furthermore, the prion protein prevents formation of spherical oligomers that normally occur during Aβ fibrillogenesis, acting as a potent inhibitor of Aβ1–42 toxicity as assessed in experiments with neuronal cell culture. These findings may provide a molecular level foundation to explain the reported protective action of the physiologically released N-terminal N1 fragment of PrP^C against Aβ neurotoxicity. They also suggest a novel approach to pharmacological intervention in Alzheimer disease.     

Interaction between Human Prion Protein and Amyloid-β (Aβ) Oligomers: ROLE OF N-TERMINAL RESIDUES*

Soluble oligomers of Aβ42 peptide are believed to play a major role in the pathogenesis of Alzheimer disease (AD). It was recently found that at least some of the neurotoxic effects of these oligomers may be mediated by specific binding to the prion protein, PrP^C, on the cell surface (Laurén, J., Gimbel, D. A., Nygaard, H. B., Gilbert, J. W., and Strittmatter, S. M. (2009) Nature 457, 1128–1132). Here we characterized the interaction between synthetic Aβ42 oligomers and the recombinant human prion protein (PrP) using two biophysical techniques: site-directed spin labeling and surface plasmon resonance. Our data indicate that this binding is highly specific for a particular conformation adopted by the peptide in soluble oligomeric species. The binding appears to be essentially identical for the Met¹²⁹ and Val¹²⁹ polymorphic forms of human PrP, suggesting that the role of PrP codon 129 polymorphism as a risk factor in AD is due to factors unrelated to the interaction with Aβ oligomers. It was also found that in addition to the previously identified ∼95–110 segment, the second region of critical importance for the interaction with Aβ42 oligomers is a cluster of basic residues at the extreme N terminus of PrP (residues 23–27). The deletion of any of these segments results in a major loss of the binding function, indicating that these two regions likely act in concert to provide a high affinity binding site for Aβ42 oligomers. This insight may help explain the interplay between the postulated protective and pathogenic roles of PrP in AD and may contribute to the development of novel therapeutic strategies as well.     

Contribution of Specific Residues of the β-Solenoid Fold to HET-s Prion Function,Amyloid Structure and Stability

The [Het-s] prion of the fungus Podospora anserina represents a good model system for studying the structure-function relationship in amyloid proteins because a high resolution solid-state NMR structure of the amyloid prion form of the HET-s prion forming domain (PFD) is available. The HET-s PFD adopts a specific β-solenoid fold with two rungs of β-strands delimiting a triangular hydrophobic core. A C-terminal loop folds back onto the rigid core region and forms a more dynamic semi-hydrophobic pocket extending the hydrophobic core. Herein, an alanine scanning mutagenesis of the HET-s PFD was conducted. Different structural elements identified in the prion fold such as the triangular hydrophobic core, the salt bridges, the asparagines ladders and the C-terminal loop were altered and the effect of these mutations on prion function, fibril structure and stability was assayed. Prion activity and structure were found to be very robust; only a few key mutations were able to corrupt structure and function. While some mutations strongly destabilize the fold, many substitutions in fact increase stability of the fold. This increase in structural stability did not influence prion formation propensity in vivo. However, if an Ala replacement did alter the structure of the core or did influence the shape of the denaturation curve, the corresponding variant showed a decreased prion efficacy. It is also the finding that in addition to the structural elements of the rigid core region, the aromatic residues in the C-terminal semi-hydrophobic pocket are critical for prion propagation. Mutations in the latter region either positively or negatively affected prion formation. We thus identify a region that modulates prion formation although it is not part of the rigid cross-β core, an observation that might be relevant to other amyloid models.     

Interaction of Amyloid Inhibitor Proteins with Amyloid Beta Peptides: Insight from Molecular Dynamics Simulations

Knowledge of the detailed mechanism by which proteins such as human αB- crystallin and human lysozyme inhibit amyloid beta (Aβ) peptide aggregation is crucial for designing treatment for Alzheimer''s disease. Thus, unconstrained, atomistic molecular dynamics simulations in explicit solvent have been performed to characterize the Aβ_17–42 assembly in presence of the αB-crystallin core domain and of lysozyme. Simulations reveal that both inhibitor proteins compete with inter-peptide interaction by binding to the peptides during the early stage of aggregation, which is consistent with their inhibitory action reported in experiments. However, the Aβ binding dynamics appear different for each inhibitor. The binding between crystallin and the peptide monomer, dominated by electrostatics, is relatively weak and transient due to the heterogeneous amino acid distribution of the inhibitor surface. The crystallin-bound Aβ oligomers are relatively long-lived, as they form more extensive contact surface with the inhibitor protein. In contrast, a high local density of arginines from lysozyme allows strong binding with Aβ peptide monomers, resulting in stable complexes. Our findings not only illustrate, in atomic detail, how the amyloid inhibitory mechanism of human αB-crystallin, a natural chaperone, is different from that of human lysozyme, but also may aid de novo design of amyloid inhibitors.     

The Metalloprotease Meprin β Generates Amino Terminal-truncated Amyloid β Peptide Species

The amyloid β (Aβ) peptide, which is abundantly found in the brains of patients suffering from Alzheimer disease, is central in the pathogenesis of this disease. Therefore, to understand the processing of the amyloid precursor protein (APP) is of critical importance. Recently, we demonstrated that the metalloprotease meprin β cleaves APP and liberates soluble N-terminal APP (N-APP) fragments. In this work, we present evidence that meprin β can also process APP in a manner reminiscent of β-secretase. We identified cleavage sites of meprin β in the amyloid β sequence of the wild type and Swedish mutant of APP at positions p1 and p2, thereby generating Aβ variants starting at the first or second amino acid residue. We observed even higher kinetic values for meprin β than BACE1 for both the wild type and the Swedish mutant APP form. This enzymatic activity of meprin β on APP and Aβ generation was also observed in the absence of BACE1/2 activity using a β-secretase inhibitor and BACE knock-out cells, indicating that meprin β acts independently of β-secretase.     

Brain Endothelial Cells Produce Amyloid β from Amyloid Precursor Protein 770 and Preferentially Secrete the O-Glycosylated Form

Deposition of amyloid β (Aβ) in the brain is closely associated with Alzheimer disease (AD). Aβ is generated from amyloid precursor protein (APP) by the actions of β- and γ-secretases. In addition to Aβ deposition in the brain parenchyma, deposition of Aβ in cerebral vessel walls, termed cerebral amyloid angiopathy, is observed in more than 80% of AD individuals. The mechanism for how Aβ accumulates in blood vessels remains largely unknown. In the present study, we show that brain endothelial cells expressed APP770, a differently spliced APP mRNA isoform from neuronal APP695, and produced Aβ40 and Aβ42. Furthermore, we found that the endothelial APP770 had sialylated core 1 type O-glycans. Interestingly, Ο-glycosylated APP770 was preferentially processed by both α- and β-cleavage and secreted into the media, suggesting that O-glycosylation and APP processing involved related pathways. By immunostaining human brain sections with an anti-APP770 antibody, we found that APP770 was expressed in vascular endothelial cells. Because we were able to detect O-glycosylated sAPP770β in human cerebrospinal fluid, this unique soluble APP770β has the potential to serve as a marker for cortical dementias such as AD and vascular dementia.     

Parallel In-register Intermolecular β-Sheet Architectures for Prion-seeded Prion Protein (PrP) Amyloids

Structures of the infectious form of prion protein (e.g. PrP^Sc or PrP-Scrapie) remain poorly defined. The prevalent structural models of PrP^Sc retain most of the native α-helices of the normal, noninfectious prion protein, cellular prion protein (PrP^C), but evidence is accumulating that these helices are absent in PrP^Sc amyloid. Moreover, recombinant PrP^C can form amyloid fibrils in vitro that have parallel in-register intermolecular β-sheet architectures in the domains originally occupied by helices 2 and 3. Here, we provide solid-state NMR evidence that the latter is also true of initially prion-seeded recombinant PrP amyloids formed in the absence of denaturants. These results, in the context of a primarily β-sheet structure, led us to build detailed models of PrP amyloid based on parallel in-register architectures, fibrillar shapes and dimensions, and other available experimentally derived conformational constraints. Molecular dynamics simulations of PrP(90–231) octameric segments suggested that such linear fibrils, which are consistent with many features of PrP^Sc fibrils, can have stable parallel in-register β-sheet cores. These simulations revealed that the C-terminal residues ∼124–227 more readily adopt stable tightly packed structures than the N-terminal residues ∼90–123 in the absence of cofactors. Variations in the placement of turns and loops that link the β-sheets could give rise to distinct prion strains capable of faithful template-driven propagation. Moreover, our modeling suggests that single PrP monomers can comprise the entire cross-section of fibrils that have previously been assumed to be pairs of laterally associated protofilaments. Together, these insights provide a new basis for deciphering mammalian prion structures.     

The ultrastructure of infectious L-type bovine spongiform encephalopathy prions constrains molecular models

Bovine spongiform encephalopathy (BSE) is a prion disease of cattle that is caused by the misfolding of the cellular prion protein (PrP^C) into an infectious conformation (PrP^Sc). PrP^C is a predominantly α-helical membrane protein that misfolds into a β-sheet rich, infectious state, which has a high propensity to self-assemble into amyloid fibrils. Three strains of BSE prions can cause prion disease in cattle, including classical BSE (C-type) and two atypical strains, named L-type and H-type BSE. To date, there is no detailed information available about the structure of any of the infectious BSE prion strains. In this study, we purified L-type BSE prions from transgenic mouse brains and investigated their biochemical and ultrastructural characteristics using electron microscopy, image processing, and immunogold labeling techniques. By using phosphotungstate anions (PTA) to precipitate PrP^Sc combined with sucrose gradient centrifugation, a high yield of proteinase K-resistant BSE amyloid fibrils was obtained. A morphological examination using electron microscopy, two-dimensional class averages, and three-dimensional reconstructions revealed two structural classes of L-type BSE amyloid fibrils; fibrils that consisted of two protofilaments with a central gap and an average width of 22.5 nm and one-protofilament fibrils that were 10.6 nm wide. The one-protofilament fibrils were found to be more abundant compared to the thicker two-protofilament fibrils. Both fibrillar assemblies were successfully decorated with monoclonal antibodies against N- and C-terminal epitopes of PrP using immunogold-labeling techniques, confirming the presence of polypeptides that span residues 100–110 to 227–237. The fact that the one-protofilament fibrils contain both N- and C-terminal PrP epitopes constrains molecular models for the structure of the infectious conformer in favour of a compact four-rung β-solenoid fold.     

Is tau ready for admission to the prion club?

Aggregation-prone proteins associated with neurodegenerative disease, such as α synuclein and β 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.     

Signal Transduction by a Fungal NOD-Like Receptor Based on Propagation of a Prion Amyloid Fold

In the fungus Podospora anserina, the [Het-s] prion induces programmed cell death by activating the HET-S pore-forming protein. The HET-s β-solenoid prion fold serves as a template for converting the HET-S prion-forming domain into the same fold. This conversion, in turn, activates the HET-S pore-forming domain. The gene immediately adjacent to het-S encodes NWD2, a Nod-like receptor (NLR) with an N-terminal motif similar to the elementary repeat unit of the β-solenoid fold. NLRs are immune receptors controlling cell death and host defense processes in animals, plants and fungi. We have proposed that, analogously to [Het-s], NWD2 can activate the HET-S pore-forming protein by converting its prion-forming region into the β-solenoid fold. Here, we analyze the ability of NWD2 to induce formation of the β-solenoid prion fold. We show that artificial NWD2 variants induce formation of the [Het-s] prion, specifically in presence of their cognate ligands. The N-terminal motif is responsible for this prion induction, and mutations predicted to affect the β-solenoid fold abolish templating activity. In vitro, the N-terminal motif assembles into infectious prion amyloids that display a structure resembling the β-solenoid fold. In vivo, the assembled form of the NWD2 N-terminal region activates the HET-S pore-forming protein. This study documenting the role of the β-solenoid fold in fungal NLR function further highlights the general importance of amyloid and prion-like signaling in immunity-related cell fate pathways.     

SMG1 Identified as a Regulator of Parkinson’s Disease-Associated alpha-Synuclein through siRNA Screening

Synucleinopathies are a broad class of neurodegenerative disorders characterized by the presence of intracellular protein aggregates containing α-synuclein protein. The aggregated α-synuclein protein is hyperphosphorylated on serine 129 (S129) compared to the unaggregated form of the protein. While the precise functional consequences of S129 hyperphosphorylation are still being clarified, numerous in vitro and in vivo studies suggest that S129 phosphorylation is an early event in α-synuclein dysfunction and aggregation. Identifying the kinases and phosphatases that regulate this critical phosphorylation event may ultimately prove beneficial by allowing pharmacological mitigation of synuclein dysfunction and toxicity in Parkinson’s disease and other synucleinopathies. We report here the development of a high-content, fluorescence-based assay to quantitate levels of total and S129 phosphorylated α-synuclein protein. We have applied this assay to conduct high-throughput loss-of-function screens with siRNA libraries targeting 711 known and predicted human kinases and 206 phosphatases. Specifically, knockdown of the phosphatidylinositol 3-kinase related kinase SMG1 resulted in significant increases in the expression of pS129 phosphorylated α-synuclein (p-syn). Moreover, SMG1 protein levels were significantly reduced in brain regions with high p-syn levels in both dementia with Lewy bodies (DLB) and Parkinson’s disease with dementia (PDD). These findings suggest that SMG1 may play an important role in increased α-synuclein pathology during the course of PDD, DLB, and possibly other synucleinopathies.