Differential Membrane Binding and Seeding of Distinct α-Synuclein Fibrillar Polymorphs.

The aggregation of the protein α-synuclein (α-Syn) leads to different synucleinopathies. We recently showed that structurally distinct fibrillar α-Syn polymorphs trigger either Parkinson's disease or multiple system atrophy hallmarks in vivo. Here, we establish a structural-molecular basis for these observations. We show that distinct fibrillar α-Syn polymorphs bind to and cluster differentially at the plasma membrane in both primary neuronal cultures and organotypic hippocampal slice cultures from wild-type mice. We demonstrate a polymorph-dependent and concentration-dependent seeding. We show a polymorph-dependent differential synaptic redistribution of α3-Na+/K+-ATPase, GluA2 subunit containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, and GluN2B-subunit containing N-methyl-D-aspartate receptors, but not GluA1 subunit containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and metabotropic glutamate receptor 5 receptors. We also demonstrate polymorph-dependent alteration in neuronal network activity upon seeded aggregation of α-Syn. Our findings bring new, to our knowledge, insight into how distinct α-Syn polymorphs differentially bind to and seed monomeric α-Syn aggregation within neurons, thus affecting neuronal homeostasis through the redistribution of synaptic proteins.

Toxic effects of amyloid fibrils on cell membranes: the importance of ganglioside GM1.

The interaction of amyloid aggregates with the cell plasma membrane is currently considered among the basic mechanisms of neuronal dysfunction in amyloid neurodegeneration. We used amyloid oligomers and fibrils grown from the yeast prion Sup35p, responsible for the specific prion trait [PSI(+)], to investigate how membrane lipids modulate fibril interaction with the membranes of cultured H-END cells and cytotoxicity. Sup35p shares no homology with endogenous mammalian polypeptide chains. Thus, the generic toxicity of amyloids and the molecular events underlying cell degeneration can be investigated without interference with analogous polypeptides encoded by the cell genome. Sup35 fibrils bound to the cell membrane without increasing its permeability to Ca(2+). Fibril binding resulted in structural reorganization and aggregation of membrane rafts, with GM1 clustering and alteration of its mobility. Sup35 fibril binding was affected by GM1 or its sialic acid moiety, but not by cholesterol membrane content, with complete inhibition after treatment with fumonisin B1 or neuraminidase. Finally, cell impairment resulted from caspase-8 activation after Fas receptor translocation on fibril binding to the plasma membrane. Our observations suggest that amyloid fibrils induce abnormal accumulation and overstabilization of raft domains in the cell membrane and provide a reasonable, although not unique, mechanistic and molecular explanation for fibril toxicity.

AAV-mediated expression of a new conformational anti-aggregated α-synuclein antibody prolongs survival in a genetic model of α-synucleinopathies.

Prion-like transmission of pathology in α-synucleinopathies like Parkinson's disease or multiple system atrophy is increasingly recognized as one potential mechanism to address disease progression. Active and passive immunotherapies targeting insoluble, aggregated α-synuclein are already being actively explored in the clinic with mixed outcomes so far. Here, we report the identification of 306C7B3, a highly selective, aggregate-specific α-synuclein antibody with picomolar affinity devoid of binding to the monomeric, physiologic protein. 306C7B3 binding is Ser129-phosphorylation independent and shows high affinity to several different aggregated α-synuclein polymorphs, increasing the likelihood that it can also bind to the pathological seeds assumed to drive disease progression in patients. In support of this, highly selective binding to pathological aggregates in postmortem brains of MSA patients was demonstrated, with no staining in samples from other human neurodegenerative diseases. To achieve CNS exposure of 306C7B3, an adeno-associated virus (AAV) based approach driving expression of the secreted antibody within the brain of (Thy-1)-[A30P]-hα-synuclein mice was used. Widespread central transduction after intrastriatal inoculation was ensured by using the AAV2HBKO serotype, with transduction being spread to areas far away from the inoculation site. Treatment of (Thy-1)-[A30P]-hα-synuclein mice at the age of 12 months demonstrated significantly increased survival, with 306C7B3 concentration reaching 3.9 nM in the cerebrospinal fluid. These results suggest that AAV-mediated expression of 306C7B3, targeting extracellular, presumably disease-propagating aggregates of α-synuclein, has great potential as a disease-modifying therapy for α-synucleinopathies as it ensures CNS exposure of the antibody, thereby mitigating the selective permeability of the blood-brain barrier.

Recovery of small infectious PrP(res) aggregates from prion-infected cultured cells.

Prion diseases are characterized by deposits of abnormal conformers of the PrP protein. Although large aggregates of proteinase K-resistant PrP (PrP(res)) are infectious, the precise relationships between aggregation state and infectivity remain to be established. In this study, we have fractionated detergent lysates from prion-infected cultured cells by differential ultracentrifugation and ultrafiltration and have characterized a previously unnoticed PrP species. This abnormal form is resistant to proteinase K digestion but, in contrast to typical aggregated PrP(res), remains in the soluble fraction at intermediate centrifugal forces and is not retained by filters of 300-kDa cutoff. Cell-based assay and inoculation to animals demonstrate that these entities are infectious. The finding that cell-derived small infectious PrP(res) aggregates can be recovered in the absence of strong in vitro denaturating treatments now gives a biological basis for investigating the role of small PrP aggregates in the pathogenicity and/or the multiplication cycle of prions.

Pericytes take up and degrade α-synuclein but succumb to apoptosis under cellular stress.

Parkinson's disease (PD) is characterised by the progressive loss of midbrain dopaminergic neurons and the presence of aggregated α-synuclein (α-syn). Pericytes and microglia, two non-neuronal cells contain α-syn in the human brain, however, their role in disease processes is poorly understood. Pericytes, found surrounding the capillaries in the brain are important for maintaining the blood-brain barrier, controlling blood flow and mediating inflammation. In this study, primary human brain pericytes and microglia were exposed to two different α-synuclein aggregates. Inflammatory responses were assessed using immunocytochemistry, cytometric bead arrays and proteome profiler cytokine array kits. Fixed flow cytometry was used to investigate the uptake and subsequent degradation of α-syn in pericytes. We found that the two α-syn aggregates are devoid of inflammatory and cytotoxic actions on human brain derived pericytes and microglia. Although α-syn did not induce an inflammatory response, pericytes efficiently take up and degrade α-syn through the lysosomal pathway but not the ubiquitin-proteasome system. Furthermore, when pericytes were exposed the ubiquitin proteasome inhibitor-MG132 and α-syn aggregates, there was profound cytotoxicity through the production of reactive oxygen species resulting in apoptosis. These results suggest that the observed accumulation of α-syn in pericytes in human PD brains likely plays a role in PD pathogenesis, perhaps by causing cerebrovascular instability, under conditions of cellular stress.

TNF-α and α-synuclein fibrils differently regulate human astrocyte immune reactivity and impair mitochondrial respiration.

Here, we examine the cellular changes triggered by tumor necrosis factor alpha (TNF-α) and different alpha-synuclein (αSYN) species in astrocytes derived from induced pluripotent stem cells. Human astrocytes treated with TNF-α display a strong reactive pro-inflammatory phenotype with upregulation of pro-inflammatory gene networks, activation of the nuclear factor κB (NF-κB) pathway, and release of pro-inflammatory cytokines, whereas those treated with high-molecular-weight αSYN fibrils acquire a reactive antigen (cross)-presenting phenotype with upregulation of major histocompatibility complex (MHC) genes and increased human leukocyte antigen (HLA) molecules at the cell surface. Surprisingly, the cell surface location of MHC proteins is abrogated by larger F110 fibrillar polymorphs, despite the upregulation of MHC genes. Interestingly, TNF-α and αSYN fibrils compete to drive the astrocyte immune reactive response. The astrocyte immune responses are accompanied by an impaired mitochondrial respiration, which is exacerbated in Parkinson's disease (PD) astrocytes. Our data provide evidence for astrocytic involvement in PD pathogenesis and reveal their complex immune reactive responses to exogenous stressors.

Novel antibodies detect additional α-synuclein pathology in synucleinopathies: potential development for immunotherapy.

BACKGROUND: Alpha-synuclein (α-Syn) aggregation is the primary characteristic of synucleinopathies including Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Immunotherapy targeting α-Syn has shown promising results in animal models of the disease. This study investigates the target specificity of three different active vaccines for pathological α-Syn aggregates found in human brain tissue from synucleinopathies. METHODS: Guinea pigs were immunised with 3 vaccines developed by United Neuroscience, and IgG fractions purified from the resulting immune sera (IGG-1, IGG-2 or IGG-3) were used to perform immunohistochemical staining of human cases of PD, DLB and MSA. The resulting immunoreactivity was compared to a commercially available α-Syn antibody from Novacastra (NOV) commonly used for diagnostic purposes. Images were captured from the substantia nigra (SN), temporal lobe, internal capsule, insular cortex and putamen and quantified for the percentage area with α-Syn immunoreactivity. Lewy bodies (LB) and Lewy neurites (LN) were further analysed in PD and DLB cases. RESULTS: Vaccine-generated antibodies detected more α-Syn pathology compared to NOV. The levels of α-Syn immunoreactivity varied between brain region and disease type with IGG-3 recognising the highest levels of α-Syn in most cases and in all brain regions that are affected early in disease progression. IGG-3 had a high recognition for glial inclusions found in MSA which are known to have a more compact conformation. Slot blot analysis confirmed the specificity of IGG-3 for native oligomers and fibrillar α-Syn. Higher levels of α-Syn were recognised by IGG-2 in cortical regions, and by IGG-3 in SN of PD and DLB cases. This was due to increased immunolabelling of LNs in these brain regions suggesting that IGG-2 and IGG-3 recognised additional α-Syn pathology compared to IGG-1 and NOV. Whether the unique binding properties of the antibodies produced in guinea pigs will translate in the clinic remains to be addressed, which is the main limitation of this study. CONCLUSIONS: These vaccines induce antibodies that bind α-Syn oligomers and aggregates in the human brain and specifically support the choice of the vaccine generating IGG-3 (i.e. UB-312) as a candidate for clinical trials for synucleinopathies.

Synthetic lipid vesicles recruit native-like aggregates and affect the aggregation process of the prion Ure2p: insights on vesicle permeabilization and charge selectivity.

The yeast prion Ure2p polymerizes into native-like fibrils, retaining the overall structure and binding properties of the soluble protein. Recently we have shown that, similar to amyloid oligomers, the native-like Ure2p fibrils and their precursor oligomers are highly toxic to cultured mammalian cells when added to the culture medium, whereas Ure2p amyloid fibrils generated by heating the native-like fibrils are substantially harmless. We show here that, contrary to the nontoxic amyloid fibrils, the toxic, native-like Ure2p assemblies induce a significant calcein release from negatively charged phosphatidylserine vesicles. A minor and less-specific effect was observed with zwitterionic phosphatidylcholine vesicles, suggesting that the toxic aggregates preferentially bind to negatively charged sites on lipid membranes. We also found that cholesterol-enriched phospholipid membranes are protected against permeabilization by native-like Ure2p assemblies. Moreover, vesicle permeabilization appears charge-selective, allowing calcium, but not chloride, influx to be monitored. Finally, we found that the interaction with phosphatidylserine membranes speeds up Ure2p polymerization into oligomers and fibrils structurally and morphologically similar to the native-like Ure2p assemblies arising in free solution, although less cytotoxic. These data suggest that soluble Ure2p oligomers and native-like fibrils, but not amyloid fibrils, interact intimately with negatively charged lipid membranes, where they allow selective cation influx.

Assembly of the asparagine- and glutamine-rich yeast prions into protein fibrils.

The proteins Ure2, Sup35 and Rnq1 from the baker's yeast have infectious properties, termed prions, at the origin of heritable and transmissible phenotypic changes. It is widely believed that prion properties arise from the assembly of Ure2p, Sup35p and Rnq1p into insoluble fibrils. Yeast prions possess regions crucial for their propagation that can be either N- or C-terminal. These regions have unusual amino acid composition. They are very rich in glutamine and asparagine residues and resemble in that to huntingtin, a protein involved in the neurodegenerative Huntington's disease. Yeast prions assembly process has been hypothesized to be the consequence of the properties of glutamines and asparagines to engage in polar protein-protein interactions, termed polar-zippers. While this can certainly occur under certain conditions, glutamine and asparagine residues can establish other kinds of interactions with a variety of amino acid residues thus mediating protein-protein interactions involved in the assembly of polypeptide chains into high molecular weight oligomers. This review details the interactions that can be established by glutamine and asparagine residues that may allow a better understanding of their role in mediating protein-protein interactions and prion propagation.

Biophysical and functional characterization of the N-terminal domain of the cat T1R1 umami taste receptor expressed in Escherichia coli.

Umami taste perception is mediated by the heterodimeric G-protein coupled receptors (GPCRs), formed by the assembly of T1R1 and T1R3 subunits. T1R1 and T1R3 subunits are class C GPCRs whose members share common structural homologies including a long N-terminal domain (NTD) linked to a seven transmembrane domain by a short cysteine-rich region. The NTD of the T1R1 subunit contains the primary binding site for umami stimuli, such as L-glutamate (L-Glu) for humans. Inosine-5'-monophosphate (IMP) binds at a location close to the opening of the T1R1-NTD "flytrap", thus creating the observed synergistic response between L-Glu and IMP. T1R1/T1R3 binding studies have revealed species-dependent differences. While human T1R1/T1R3 is activated specifically by L-Glu, the T1R1/T1R3 in other species is a broadly tuned receptor, sensitive to a range of L-amino acids. Because domestic cats are obligate carnivores, they display strong preferences for some specific amino acids. To better understand the structural basis of umami stimuli recognition by non-human taste receptors, we measured the binding of selected amino acids to cat T1R1/T1R3 (cT1R1/cT1R3) umami taste receptor. For this purpose, we expressed cT1R1-NTD in bacteria as inclusion bodies. After purification, refolding of the protein was achieved. Circular dichroism spectroscopic studies revealed that cT1R1-NTD was well renatured with evidence of secondary structures. Using size-exclusion chromatography coupled to light scattering, we found that the cT1R1-NTD behaves as a monomer. Ligand binding quantified by intrinsic tryptophan fluorescence showed that cT1R1-NTD is capable of binding L-amino acids with Kd values in the micromolar range. We demonstrated that IMP potentiates L-amino acid binding onto renatured cT1R1-NTD. Interestingly, our results revealed that IMP binds the extracellular domain in the absence of L-amino acids. Thus, this study demonstrates that the feasibility to produce milligram quantities of cT1R1-NTD for functional and structural studies.

Mammalian prions: tracking the infectious entities.

Protein misfolding is central to the pathogenesis of several neurodegenerative disorders. Among these disorders, prion diseases are unique because they are transmissible. The conversion of the host-encoded GPI-anchored PrP protein into a structurally altered form is crucially associated with the infectious and neurotoxic properties of the resulting abnormal PrP. Many lines of evidence indicate that distinct aggregated forms with different size and protease resistance are produced during prion multiplication. The recent isolation of various subsets of abnormal PrP, along with the improved biochemical tools and infectivity detection assays have shed light on the diversity of abnormal PrP protein and may give insights into the features of the more infectious subsets of abnormal PrP.

Qualitative and quantitative multiplexed proteomic analysis of complex yeast protein fractions that modulate the assembly of the yeast prion Sup35p.

BACKGROUND: The aggregation of the baker's yeast prion Sup35p is at the origin of the transmissible [PSI(+)] trait. We and others have shown that molecular chaperones modulate Sup35p aggregation. However, other protein classes might be involved in [PSI(+)] formation. RESULTS: We designed a functional proteomic study that combines two techniques to identify modulators of Sup35p aggregation and describe the changes associated to [PSI(+)] formation. The first allows measuring the effect of fractionated Saccharomyces cerevisiae cytosolic extracts from [PSI(+)] and [psi(-)] yeast cells on Sup35p assembly. The second is a multiplex qualitative and quantitative comparison of protein composition of active and inactive fractions using a gel-free and label-free LC-MS approach. We identify changes in proteins involved in translation, folding, degradation, oxido-reduction and metabolic processes. CONCLUSION: Our functional proteomic study provides the first inventory list of over 300 proteins that directly or indirectly affect Sup35p aggregation and [PSI(+)] formation. Our results highlight the complexity of the cellular changes accompanying [PSI(+)] formation and pave the way for in vitro studies aimed to document the effect of individual and/or combinations of proteins identified here, susceptible of affecting Sup35p assembly.

A simple, versatile and sensitive cell-based assay for prions from various species.

Detection and quantification of prion infectivity is a crucial step for various fundamental and applied aspects of prion research. Identification of cell lines highly sensitive to prion infection led to the development of cell-based titration procedures aiming at replacing animal bioassays, usually performed in mice or hamsters. However, most of these cell lines are only permissive to mouse-adapted prions strains and do not allow titration of prions from other species. In this study, we show that epithelial RK13, a cell line permissive to mouse and bank vole prion strains and to natural prion agents from sheep and cervids, enables a robust and sensitive detection of mouse and ovine-derived prions. Importantly, the cell culture work is strongly reduced as the RK13 cell assay procedure designed here does not require subcultivation of the inoculated cultures. We also show that prions effectively bind to culture plastic vessel and are quantitatively detected by the cell assay. The possibility to easily quantify a wider range of prions, including rodent experimental strains but also natural agents from sheep and cervids, should prompt the spread of cell assays for routine prion titration and lead to valuable information in fundamental and applied studies.

Molecular chaperones and the assembly of the prion Ure2p in vitro.

The protein Ure2 from Saccharomyces cerevisiae possesses prion properties at the origin of the [URE3] trait. In vivo, a high molecular weight form of inactive Ure2p is associated to [URE3]. The faithful and continued propagation of [URE3]is dependent on the expression levels of molecular chaperones from the Hsp100, -70, and -40 families; however, so far, their role is not fully documented. Here we investigate the effects of molecular chaperones from the Hsp40, Hsp70, Hsp90, and Hsp100 families and the chaperonin CCT/Tric on the assembly of full-length Ure2p. We show that Hsp104p greatly stimulates Ure2p aggregation, whereas Ssa1p, Ydj1p, Sis1p, and Hsp82p inhibit aggregation to different extents. The nature of the high molecular weight Ure2p species that forms in the presence of the different molecular chaperones and their nucleotide dependence is described. We show that Hsp104p favors the aggregation of Ure2p into non-fibrillar high molecular weight particles, whereas Ssa1p, Ydj1p, Sis1p, and Hsp82p sequester Ure2p in spherical oligomers. Using fluorescently labeled full-length Ure2p and Ure2p-(94-354) and fluorescence polarization, we show that Ssa1p binding to Ure2p is ATP-dependent, whereas that of Hsp104p is not. We also show that Ssa1p preferentially interacts with the N-terminal domain of Ure2p that is critical for prion propagation, whereas Ydj1p preferentially interacts with the C-terminal domain of the protein, and we discuss the significance of this observation. Finally, the affinities of Ssa1p, Ydj1p, and Hsp104p for Ure2p are determined. Our in vitro observations bring new insight into the mechanism by which molecular chaperones influence the propagation of [URE3].

The yeast prion Ure2p native-like assemblies are toxic to mammalian cells regardless of their aggregation state.

The yeast prion Ure2p assembles in vitro into oligomers and fibrils retaining the alpha-helix content and binding properties of the soluble protein. Here we show that the different forms of Ure2p native-like assemblies (dimers, oligomers, and fibrils) are similarly toxic to murine H-END cells when added to the culture medium. Interestingly, the amyloid fibrils obtained by heat treatment of the toxic native-like fibrils appear harmless. Moreover, the Ure2p C-terminal domain, lacking the N-terminal segment necessary for aggregation but containing the glutathione binding site, is not cytotoxic. This finding strongly supports the idea that Ure2p toxicity depends on the structural properties of the flexible N-terminal prion domain and can therefore be considered as an inherent feature of the protein, unrelated to its aggregation state but rather associated with a basic toxic fold shared by all of the Ure2p native-like assemblies. Indeed, the latter are able to interact with the cell surface, leading to alteration of calcium homeostasis, membrane permeabilization, and oxidative stress, whereas the heat-treated amyloid fibrils do not. Our results support the idea of a general mechanism of toxicity of any protein/peptide aggregate endowed with structural features, making it able to interact with cell membranes and to destabilize them. This evidence extends the widely accepted view that the toxicity by protein aggregates is restricted to amyloid prefibrillar aggregates and provides new insights into the mechanism by which native-like oligomers compromise cell viability.

Structure of the prion Ure2p in protein fibrils assembled in vitro.

The Ure2 protein from the yeast Saccharomyces cerevisiae has prion properties. In vitro and at neutral pH, soluble Ure2p spontaneously forms long, straight, insoluble protein fibrils. Two models have been proposed to account for the assembly of Ure2p into protein fibrils. The "amyloid backbone" model postulates that a segment ranging from 40 to 70 amino acids in the flexible N-terminal domain from different Ure2p molecules forms a parallel superpleated beta-structure running along the fibrils. The second model hypothesizes that assembly of full-length Ure2p is driven by limited conformational rearrangements and non-native inter- and/or intramolecular interactions between Ure2p monomers. Here, we performed a cysteine scan on residues located in the N- and C-terminal parts of Ure2p to determine whether these domains interact. Amino acid sequences centered around residue 6 in the N-terminal domain of Ure2p and residue 137 in the C-terminal moiety interacted at least transiently via intramolecular interactions. We documented the assembly properties of a Ure2p variant in which a disulfide bond was established between the N- and C-terminal domains and showed that it possesses assembly properties indistinguishable from those of wild-type Ure2p. We probed the structure of Ure2pC6C137 within the fibrils and demonstrate that the polypeptide is in a conformation similar to that of its soluble assembly-competent state. Our results constitute the first structural characterization of the N-terminal domain of Ure2p in both its soluble assembly-competent and fibrillar forms. Our data indicate that the flexibility of the N-terminal domain and conformational changes within this domain are essential for fibril formation and provide new insight into the conformational rearrangements that lead to the assembly of Ure2p into fibrils and the propagation of the [URE3] phenotype in yeast.