Clustered negative charges on the lipid membrane surface induce beta-sheet formation of prion protein fragment 106-126

The conformational conversion of prion protein (PrP) from an alpha-helix-rich normal cellular isoform (PrPC) to a beta-sheet-rich pathogenic isoform (PrPSc) is a key event in the development of prion diseases, and it takes place in caveolae, cavelike invaginations of the plasma membrane. A peptide homologous to residues 106-126 of human PrP (PrP106-126) is known to share several properties with PrPSc, e.g., the capability to form a beta-sheet and toxicity against PrPC-expressing cells. PrP106-126 is thus expected to represent a segment of PrP that is involved in the formation of PrPSc. We have examined the effect of lipid membranes containing negatively charged ganglioside, an important component of caveolae, on the secondary structure of PrP106-126 by circular dichroism. The peptide forms an alpha-helical or a beta-sheet structure on the ganglioside-containing membranes. The beta-sheet content increases with an increase of the peptide:lipid ratio, indicating that the beta-sheet formation is linked with self-association of the positively charged peptide on the negatively charged membrane surface. Analogous beta-sheet formation is also induced by membranes composed of negatively charged and neutral glycerophospholipids with high and low melting temperatures, respectively, in which lateral phase separation and clustering of negatively charged lipids occur as shown by Raman spectroscopy. Since ganglioside-containing membranes also exhibit lateral phase separation, clustered negative charges are concluded to be responsible for the beta-sheet formation of PrP106-126. In caveolae, clustered ganglioside molecules are likely to interact with the residue 106-126 region of PrPC to promote the PrPC-to-PrPSc conversion.     

The role of prion peptide structure and aggregation in toxicity and membrane binding

Prion diseases are neurodegenerative disorders associated with a conformational change in the normal cellular isoform of the prion protein, PrP(C), to an abnormal scrapie isoform, PrP(SC). Unlike the alpha-helical PrP(C), the protease-resistant core of PrP(SC) is predominantly beta-sheet and possesses a tendency to polymerize into amyloid fibrils. We performed experiments with two synthetic human prion peptides, PrP(106-126) and PrP(127-147), to determine how peptide structure affects neurotoxicity and protein-membrane interactions. Peptide solutions possessing beta-sheet and amyloid structures were neurotoxic to PC12 cells in vitro and bound with measurable affinities to cholesterol-rich phospholipid membranes at ambient conditions, but peptide solutions lacking stable beta-sheet structures and amyloid content were nontoxic and possessed less than one tenth of the binding affinities of the amyloid-containing peptides. Regardless of structure, the peptide binding affinities to cholesterol-depleted membranes were greatly reduced. These results suggest that the beta-sheet and amyloid structures of the prion peptides give rise to their toxicity and membrane binding affinities and that membrane binding affinity, especially in cholesterol-rich environments, may be related to toxicity. Our results may have significance in understanding the role of the fibrillogenic cerebral deposits associated with some of the prion diseases in neurodegeneration and may have implications for other amyloidoses.     

The Hydrophobic Core Sequence Modulates the Neurotoxic and Secondary Structure Properties of the Prion Peptide 106-126

The neurodegeneration seen in spongiform encephalopathies is believed to be mediated by protease-resistant forms of the prion protein (PrP). A peptide encompassing residues 106-126 of human PrP has been shown to be neurotoxic in vitro. The neurotoxicity of PrP106-126 appears to be dependent upon its adoption of an aggregated fibril structure. To examine the role of the hydrophobic core, AGAAAAGA, on PrP106-126 toxicity, we performed structure-activity analyses by substituting two or more hydrophobic residues for the hydrophilic serine residue to decrease its hydrophobicity. A peptide with a deleted alanine was also synthesized. We found all the peptides except the deletion mutant were no longer toxic on mouse cerebellar neuronal cultures. Circular dichroism analysis showed that the nontoxic PrP peptides had a marked decrease in beta-sheet structure. In addition, the mutants had alterations in aggregability as measured by turbidity, Congo red binding, and fibril staining using electron microscopy. These data show that the hydrophobic core sequence is important for PrP106-126 toxicity probably by influencing its assembly into a neurotoxic structure. The hydrophobic sequence may similarly affect aggregation and toxicity observed in prion diseases.     

PrP(106-126) activates neuronal intracellular kinases and Egr1 synthesis through activation of NADPH-oxidase independently of PrPc

Prion diseases are characterised by severe neural lesions linked to the presence of an abnormal protease-resistant isoform of cellular prion protein (PrPc). The peptide PrP(106-126) is widely used as a model of neurotoxicity in prion diseases. Here, we examine in detail the intracellular signalling cascades induced by PrP(106-126) in cortical neurons and the participation of PrPc. We show that PrP(106-126) induces the activation of subsets of intracellular kinases (e.g., ERK1/2), early growth response 1 synthesis and induces caspase-3 activity, all of which are mediated by nicotinamide adenine dinucleotide phosphate hydrogen-oxidase activity and oxidative stress. However, cells lacking PrPc are similarly affected after peptide exposure, and this questions the involvement of PrPc in these effects.     

PrP106-126 amide causes the semi-penetrated poration in the supported lipid bilayers

A major hallmark of prion diseases is the cerebral amyloid accumulation of the pathogenic PrP(Sc), an abnormally misfolded, protease-resistant, and beta-sheet rich protein. PrP106-126 is the key domain responsible for the conformational conversion and aggregation of PrP. It shares important physicochemical characteristics with PrP(Sc) and presents similar neurotoxicity as PrP(Sc). By combination of fluorescence polarization, dye release assay and in situ time-lapse atomic force microscopy (AFM), we investigated the PrP106-126 amide interacting with the large unilamellar vesicles (LUVs) and the supported lipid bilayers (SLBs). The results suggest that the interactions involve a poration-mediated process: firstly, the peptide binding results in the formation of pores in the membranes, which penetrate only half of the membranes; subsequently, PrP106-126 amide undergoes the poration-mediated diffusion in the SLBs, represented by the formation and expansion of the flat high-rise domains (FHDs). The possible mechanisms of the interactions between PrP106-126 amide and lipid membranes are proposed based on our observations.     

Amidation and structure relaxation abolish the neurotoxicity of the prion peptide PrP106-126 in vivo and in vitro

One of the major pathological hallmarks of transmissible spongiform encephalopathies (TSEs) is the accumulation of a pathogenic (scrapie) isoform (PrP(Sc)) of the cellular prion protein (PrP(C)) primarily in the central nervous system. The synthetic prion peptide PrP106-126 shares many characteristics with PrP(Sc) in that it shows PrP(C)-dependent neurotoxicity both in vivo and in vitro. Moreover, PrP106-126 in vitro neurotoxicity has been closely associated with the ability to form fibrils. Here, we studied the in vivo neurotoxicity of molecular variants of PrP106-126 toward retinal neurons using electroretinographic recordings in mice after intraocular injections of the peptides. We found that amidation and structure relaxation of PrP106-126 significantly reduced the neurotoxicity in vivo. This was also found in vitro in primary neuronal cultures from mouse and rat brain. Thioflavin T binding studies showed that amidation and structure relaxation significantly reduced the ability of PrP106-126 to attain fibrillar structures in physiological salt solutions. This study hence supports the assumption that the neurotoxic potential of PrP106-126 is closely related to its ability to attain secondary structure.     

Tetracycline affects abnormal properties of synthetic PrP peptides and PrP(Sc) in vitro

Prion diseases are characterized by the accumulation of altered forms of the prion protein (termed PrP(Sc)) in the brain. Unlike the normal protein, PrP(Sc) isoforms have a high content of beta-sheet secondary structure, are protease-resistant, and form insoluble aggregates and amyloid fibrils. Evidence indicates that they are responsible for neuropathological changes (i.e. nerve cell degeneration and glial cell activation) and transmissibility of the disease process. Here, we show that the antibiotic tetracycline: (i) binds to amyloid fibrils generated by synthetic peptides corresponding to residues 106-126 and 82-146 of human PrP; (ii) hinders assembly of these peptides into amyloid fibrils; (iii) reverts the protease resistance of PrP peptide aggregates and PrP(Sc) extracted from brain tissue of patients with Creutzfeldt-Jakob disease; (iv) prevents neuronal death and astrocyte proliferation induced by PrP peptides in vitro. NMR spectroscopy revealed several through-space interactions between aromatic protons of tetracycline and side-chain protons of Ala(117-119), Val(121-122) and Leu(125) of PrP 106-126. These properties make tetracycline a prototype of compounds with the potential of inactivating the pathogenic forms of PrP.     

Oxidation reduces the fibrillation but not the neurotoxicity of the prion peptide PrP106-126

There is increasing evidence that soluble oligomers of misfolded protein may play a role in the pathogenesis of protein misfolding diseases including the transmissible spongiform encephalopathies (TSE) where the protein involved is the prion protein, PrP. The effect of oxidation on fibrillation tendency and neurotoxicity of different molecular variants of the prion peptide PrP106-126 was investigated. It was found that methionine oxidation significantly reduced amyloid fibril formation and proteinase K resistance, but it did not reduce (but rather increase slightly) the neurotoxicity of the peptides in vivo (electroretinography after intraocular injections in mice) and in vitro (in primary neuronal cultures). We furthermore found that the bovine variant of PrP106-126, containing only one methionine residue, showed both reduced fibril forming capacity and in vivo and in vitro neurotoxicity. The findings imply (I) that there is not a simple relation between the formation of amyloid fibrils and neurotoxicity of PrP106-126 derived peptides, (II) that putative, soluble, non-amyloid protofibrils, presumed to be present in increased proportions in oxidized PrP106-126, could play a role in the pathogenesis of TSE and III) that the number of methionine residues in the PrP106-126 peptide seems to have a pivotal role in determining the physical and biological properties of PrP106-126.     

Zinc, copper, and carnosine attenuate neurotoxicity of prion fragment PrP106-126

Prion diseases are progressive neurodegenerative diseases that are associated with the conversion of normal cellular prion protein (PrP(C)) to abnormal pathogenic prion protein (PrP(SC)) by conformational changes. Prion protein is a metal-binding protein that is suggested to be involved in metal homeostasis. We investigated here the effects of trace elements on the conformational changes and neurotoxicity of synthetic prion peptide (PrP106-126). PrP106-126 exhibited the formation of β-sheet structures and enhanced neurotoxicity during the aging process. The co-existence of Zn(2+) or Cu(2+) during aging inhibited β-sheet formation by PrP106-126 and attenuated its neurotoxicity on primary cultured rat hippocampal neurons. Although PrP106-126 formed amyloid-like fibrils as observed by atomic force microscopy, the height of the fibers was decreased in the presence of Zn(2+) or Cu(2+). Carnosine (β-alanyl histidine) significantly inhibited both the β-sheet formation and the neurotoxicity of PrP106-126. Our results suggested that Zn(2+) and Cu(2+) might be involved in the pathogenesis of prion diseases. It is also possible that carnosine might become a candidate for therapeutic treatments for prion diseases.     

Contribution of two conserved glycine residues to fibrillogenesis of the 106-126 prion protein fragment. Evidence that a soluble variant of the 106-126 peptide is neurotoxic

The fibrillogenic peptide corresponding to the residues 106-126 of the prion protein sequence (PrP 106-126) is largely used to explore the neurotoxic mechanisms underlying the prion disease. However, whether the neuronal toxicity of PrP 106-126 is caused by a soluble or fibrillar form of this peptide is still unknown. The aim of this study was to correlate the structural state of this peptide with its neurotoxicity. Here we show that the two conserved Gly114 and Gly119 residues, in force of their intrinsic flexibility, prevent the peptide assuming a structured conformation, favouring its aggregation in amyloid fibrils. The substitution of both Gly114 and Gly119 with alanine residues (PrP 106-126 AA mutated peptide) reduces the flexibility of this prion fragment and results in a soluble, beta-structured peptide. Moreover, PrP 106-126 AA fragment was highly toxic when incubated with neuroblastoma cells, likely behaving as a neurotoxic protofibrillar intermediate of the wild-type PrP 106-126. These data further confirm that the fibrillar aggregation is not necessary for the induction of the toxic effects of PrP 106-126.     

The neurotoxic prion peptide fragment PrP(106-126) is a chemotactic agonist for the G protein-coupled receptor formyl peptide receptor-like 1

Prion diseases are transmissible and fatal neurodegenerative disorders which involve infiltration and activation of mononuclear phagocytes at the brain lesions. A 20-aa acid fragment of the human cellular prion protein, PrP(106-126), was reported to mimic the biological activity of the pathologic isoform of prion and activates mononuclear phagocytes. The cell surface receptor(s) mediating the activity of PrP(106-126) is unknown. In this study, we show that PrP(106-126) is chemotactic for human monocytes through the use of a G protein-coupled receptor formyl peptide receptor-like 1 (FPRL1), which has been reported to interact with a diverse array of exogenous or endogenous ligands. Upon stimulation by PrP(106-126), FPRL1 underwent a rapid internalization and, furthermore, PrP(106-126) enhanced monocyte production of proinflammatory cytokines, which was inhibited by pertussis toxin. Thus, FPRL1 may act as a "pattern recognition" receptor that interacts with multiple pathologic agents and may be involved in the proinflammatory process of prion diseases.     

Small stress molecules inhibit aggregation and neurotoxicity of prion peptide 106-126

In prion diseases, the posttranslational modification of host-encoded prion protein PrP^c yields a high β-sheet content modified protein PrP^sc, which further polymerizes into amyloid fibrils. PrP106-126 initiates the conformational changes leading to the conversion of PrP^c to PrP^sc. Molecules that can defunctionalize such peptides can serve as a potential tool in combating prion diseases. In microorganisms during stressed conditions, small stress molecules (SSMs) are formed to prevent protein denaturation and maintain protein stability and function. The effect of such SSMs on PrP106-126 amyloid formation is explored in the present study using turbidity, atomic force microscopy (AFM), and cellular toxicity assay. Turbidity and AFM studies clearly depict that the SSMs—ectoine and mannosylglyceramide (MGA) inhibit the PrP106-126 aggregation. Our study also connotes that ectoine and MGA offer strong resistance to prion peptide-induced toxicity in human neuroblastoma cells, concluding that such molecules can be potential inhibitors of prion aggregation and toxicity.     

Aggregation of PrP106–126 on surfaces of neutral and negatively charged membranes studied by molecular dynamics simulations

Prion diseases are neurodegenerative disorders characterized by the aggregation of an abnormal form of prion protein. The interaction of prion protein and cellular membrane is crucial to elucidate the occurrence and development of prion diseases. Its fragment, residues 106–126, has been proven to maintain the pathological properties of misfolded prion and was used as a model peptide. In this study, explicit solvent molecular dynamics (MD) simulations were carried out to investigate the adsorption, folding and aggregation of PrP106–126 with different sizes (2-peptides, 4-peptides and 6-peptides) on the surface of both pure neutral POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) and negatively charged POPC/POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol) (3:1) lipids. MD simulation results show that PrP106–126 display strong affinity with POPC/POPG but does not interact with pure POPC. The positively charged and polar residues participating hydrogen bonding with membrane promote the adsorption of PrP106–126. The presence of POPC and POPC/POPG exert limited influence on the secondary structures of PrP106–126 and random coil structures are predominant in all simulation systems. Upon the adsorption on the POPC/POPG surface, the aggregation states of PrP106–126 have been changed and more small oligomers were observed. This work provides insights into the interactions of PrP106–126 and membranes with different compositions in atomic level, which expand our understanding the role membrane plays in the development of prion diseases. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy.     

Heat shock protein 104 inhibited the fibrillization of prion peptide 106-126 and disassembled prion peptide 106-126 fibrils in vitro

Amyloid-like fibrils have been associated with the pathogenesis of human prion diseases. Prion peptide of aa 106-126 (PrP106-126) exhibits many PrP(Sc)-like biochemical features, forming amyloid-like fibrils in vitro. Here, we found that the recombinant yeast-derived molecular chaperon Hsp104 inhibited significantly the fibril assembly of the synthetic PrP106-126 peptide by dynamic ThT assays in vitro. EM assays revealed almost no fibril-like structure after incubation of the synthetic PrP106-126 peptides with Hsp104 for 12h. Circular dichroism assays identified that treatment of Hsp104 shifted the secondary structure of PrP106-126 fibrils from β-sheet to a random coil. MTT tests confirmed that interaction of PrP106-126 with Hsp104 maintained the toxicity of PrP106-126 on human neuroblastoma cell line SK-N-SH. Additionally, Hsp104 was able to disassemble the mature PrP106-126 fibrils in vitro, leading to recovering the cytotoxicity of PrP106-126 on SK-N-SH cells. Our study provides the molecular evidences that the yeast-derived Hsp104 can interfere in the fibril assembly and disassembly of human PrP106-126 segment.     

Prion peptide 106-126 modulates the aggregation of cellular prion protein and induces the synthesis of potentially neurotoxic transmembrane PrP.

In infectious and familial prion disorders, neurodegeneration is often seen without obvious deposits of the scrapie prion protein (PrP(Sc)), the principal cause of neuronal death in prion disorders. In such cases, neurotoxicity must be mediated by alternative pathways of cell death. One such pathway is through a transmembrane form of PrP. We have investigated the relationship between intracellular accumulation of prion protein aggregates and the consequent up-regulation of transmembrane prion protein in a cell model. Here, we report that exposure of neuroblastoma cells to the prion peptide 106-126 catalyzes the aggregation of cellular prion protein to a weakly proteinase K-resistant form and induces the synthesis of transmembrane prion protein, the proposed mediator of neurotoxicity in certain prion disorders. The N terminus of newly synthesized transmembrane prion protein is cleaved spontaneously on the cytosolic face of the endoplasmic reticulum, and the truncated C-terminal fragment accumulates on the cell surface. Our results suggest that neurotoxicity in prion disorders is mediated by a complex pathway involving transmembrane prion protein and not by deposits of aggregated and proteinase K-resistant PrP alone.     

Cellular uptake of the prion protein fragment PrP106-126 in vitro

The aetiological agent of prion disease is proposed to be an aberrant isoform of the cell surface glycoprotein known as the prion protein (PrP^c). This pathological isoform (PrP^Sc) is abnormally deposited in the extracellular space of diseased CNS. Neurodegeneration in these disease has been shown to be associated with accumulation of PrP^Sc in affected tissue. To investigate the possible uptake mechanisms that may be required for PrP^Sc-induced neurodegeneration we studied the cellular trafficking of the neurotoxic fragment, PrP106-126. We were able to detect, by fluorescence microscopy, PrP106-126 inclusions in murine neurones, astrocytes and microglia in vitro. These inclusions were abundant after 24 hour exposure and still present 48h post-exposure. Shorter exposure times yielded only occasional cells with inclusions. Large extracellular aggregates of PrP106-126 could also be detected, which appeared in a time dependent manner. The appearance of inclusions or aggregates was not dependent on PrP^c expression as determined by exposure of peptides from PrP-null mice. Using transmission electron microscopy and gold particle detection, positively labelled osmiophilic inclusions of peptide could be detected in the cytoplasm of exposed cells. These results demonstrate that cultured cells are capable of sequestering PrP106-126 and may indicate uptake pathways for PrP^Sc in various cell types. Toxicity of PrP106-126 may thus be mediated via a sequestration pathway that is not effective for this peptide in PrP-null cells.     

Prion Protein Peptide Neurotoxicity Can Be Mediated by Astrocytes

A peptide based on amino acids 106-126 of the sequence of human prion protein (PrP106-126) is neurotoxic in culture. A role for astrocytes mediating PrP106-126 toxicity was investigated. The toxicity of PrP106-126 to cerebellar cell cultures was reduced by aminoadipate, a gliotoxin. Normally, PrP106-126 is not toxic to cultures containing neurones deficient in the cellular isoform of prion protein (PrPc). However, PrP106-126 was toxic to cerebellar cells derived from Prnp(0/0) mice (deficient in PrPc expression) when those cerebellar cells were cocultured with astrocytes. This toxicity was found to occur only in the presence of PrPc-positive astrocytes and to be mediated by glutamate. Furthermore, PrPc-positive astrocytes were shown to protect Prnp(0/0) cerebellar cells from glutamate toxicity. This effect could be inhibited by PrP106-126. PrP106-126 did not enhance the toxicity of glutamate to neurones directly. When cerebellar cells were cocultured with astrocytes, the neurones became dependent on astrocytes for protection from glutamate toxicity and expressed an increased sensitivity to glutamate. In such a system, the protective effects of astrocytes against glutamate toxicity to neurones were inhibited by PrP106-126, resulting in a greater reduction in neuronal survival than would have been caused by PrP106-126 when astrocytes were not present. This new model provides a possible mechanism by which the gliosis in prion disease may accelerate the neurodegeneration seen in the later stages of the disease.     

CD36 participates in PrP(106-126)-induced activation of microglia

Microglial activation is a characteristic feature of the pathogenesis of prion diseases. The molecular mechanisms that underlie prion-induced microglial activation are not very well understood. In the present study, we investigated the role of the class B scavenger receptor CD36 in microglial activation induced by neurotoxic prion protein (PrP) fragment 106-126 (PrP(106-126)). We first examined the time course of CD36 mRNA expression upon exposure to PrP(106-126) in BV2 microglia. We then analyzed different parameters of microglial activation in PrP(106-126)-treated cells in the presence or not of anti-CD36 monoclonal antibody (mAb). The cells were first incubated for 1 h with CD36 monoclonal antibody to block the CD36 receptor, and were then treated with neurotoxic prion peptides PrP(106-126). The results showed that PrP(106-126) treatment led to a rapid yet transitory increase in the mRNA expression of CD36, upregulated mRNA and protein levels of proinflammatory cytokines (IL-1β, IL-6 and TNF-α), increased iNOS expression and nitric oxide (NO) production, stimulated the activation of NF-κB and caspase-1, and elevated Fyn activity. The blockade of CD36 had no effect on PrP(106-126)-stimulated NF-κB activation and TNF-α protein release, abrogated the PrP(106-126)-induced iNOS stimulation, downregulated IL-1β and IL-6 expression at both mRNA and protein levels as well as TNF-α mRNA expression, decreased NO production and Fyn phosphorylation, reduced caspase-1 cleavage induced by moderate PrP(106-126)-treatment, but had no effect on caspase-1 activation after treatment with a high concentration of PrP(106-126). Together, these results suggest that CD36 is involved in PrP(106-126)-induced microglial activation and that the participation of CD36 in the interaction between PrP(106-126) and microglia may be mediated by Src tyrosine kinases. Our findings provide new insights into the mechanisms underlying the activation of microglia by neurotoxic prion peptides and open perspectives for new therapeutic strategies for prion diseases by modulation of CD36 signaling.     

Imaging Flow Cytometry Illuminates New Dimensions of Amyloid Peptide-Membrane Interactions

Membrane interactions of amyloidogenic proteins constitute central determinants both in protein aggregation as well as in amyloid cytotoxicity. Most reported studies of amyloid peptide-membrane interactions have employed model membrane systems combined with application of spectroscopy methods or microscopy analysis of individual binding events. Here, we applied for the first time, to our knowledge, imaging flow cytometry for investigating interactions of representative amyloidogenic peptides, namely, the 106–126 fragment of prion protein (PrP(106–126)) and the human islet amyloid polypeptide (hIAPP), with giant lipid vesicles. Imaging flow cytometry was also applied to examine the inhibition of PrP(106–126)-membrane interactions by epigallocatechin gallate, a known modulator of amyloid peptide aggregation. We show that imaging flow cytometry provided comprehensive population-based statistical information upon morphology changes of the vesicles induced by PrP(106–126) and hIAPP. Specifically, the experiments reveal that both PrP(106–126) and hIAPP induced dramatic transformations of the vesicles, specifically disruption of the spherical shapes, reduction of vesicle circularity, lobe formation, and modulation of vesicle compactness. Interesting differences, however, were apparent between the impact of the two peptides upon the model membranes. The morphology analysis also showed that epigallocatechin gallate ameliorated vesicle disruption by PrP(106–126). Overall, this study demonstrates that imaging flow cytometry provides powerful means for disclosing population-based morphological membrane transformations induced by amyloidogenic peptides and their inhibition by aggregation modulators.