Internalization of PrP106-126 by the formyl-peptide-receptor-like-1 in glial cells

Recent studies suggest that the formyl-peptide-receptor-like-1 (FPRL1) plays an essential role in inflammatory responses in the host defence mechanisms and neurodegenerative disorders. Furthermore, it may be involved in proinflammatory processes of prion diseases. However, little is known about the induction and regulation of PrP106-126-induced receptor endocytosis. We have thus analysed whether PrP106-126 increases the activity of phospholipase D (PLD) via FPRL1, an enzyme involved in the regulation of the secretion, endocytosis and receptor signalling, in glial cells. PLD activity was determined using a transphosphatidylation assay and the internalization of PrP106-126, and FPRL1 was assessed by fluorescence microscopy and quantified by ELISA. We could show that PLD is activated by PrP106-126 both in astrocytes and microglia, and moreover that PrP106-126 is rapidly internalized via FPRL1 in astrocytes and microglia cells. The determination of receptor activity by extracellular signal-regulated kinases 1/2 phosphorylation and cAMP level measurement verified the PrP106-126-induced activation of FPRL1. FPRL1-mediated PrP106-126 uptake was blocked by the receptor antagonist chenodeoxycholic acid. These studies indicate the involvement of FPRL1-mediated cellular signalling in PrP106-126-endocytosis and may allow the development of therapeutic agents interfering with prion uptake and/or PLD function, using either PLD or the FPRL1 as a possible pharmaceutical target.     

A new staphylococcal anti-inflammatory protein that antagonizes the formyl peptide receptor-like 1

Bacteria have developed mechanisms to escape the first line of host defense, which is constituted by the recruitment of phagocytes to the sites of bacterial invasion. We previously described the chemotaxis inhibitory protein of Staphylococcus aureus, a protein that blocks the activation of neutrophils via the formyl peptide receptor (FPR) and C5aR. We now describe a new protein from S. aureus that impaired the neutrophil responses to FPR-like1 (FPRL1) agonists. FPRL1 inhibitory protein (FLIPr) inhibited the calcium mobilization in neutrophils stimulated with MMK-1, WKYMVM, prion-protein fragment PrP(106-126), and amyloid beta(1-42). Stimulation with low concentrations of fMLP was partly inhibited. Directed migration was also completely prevented toward MMK-1 and partly toward fMLP. Fluorescence-labeled FLIPr efficiently bound to neutrophils, monocytes, B cells, and NK cells. HEK293 cells transfected with human C5aR, FPR, FPRL1, and FPRL2 clearly showed that FLIPr directly bound to FPRL1 and, at higher concentrations, also to FPR but not to C5aR and FPRL2. FLIPr can reveal unknown inflammatory ligands crucial during S. aureus infections. As a novel described FPRL1 antagonist, it might lead to the development of therapeutic agents in FPRL1-mediated inflammatory components of diseases such as systemic amyloidosis, Alzheimer's, and prion disease.     

Pleiotropic roles of formyl peptide receptors

FPR and FPRL1 belong to the seven-transmembrane, G protein-coupled chemoattractant receptor superfamily. Because of their capacity to interact with bacterial chemotactic formylated peptides, these receptors are thought to play a role in host defense against microbial infection. Recently, a variety of novel agonists have been identified for these receptors, including several host-derived endogenous molecules that are involved in proinflammatory responses. Most notably is the use of FPRL1 by at least three amyloidogenic protein and peptide ligands, the serum amyloid A (SAA), the 42 amino acid form of beta amyloid (Abeta(42)), and the prion peptide PrP106-126, to chemoattract and activate human phagocytic leukocytes. These new findings have greatly expanded the functional scope of the formyl peptide receptors and call for more in-depth investigation of the role of these receptors in pathophysiological conditions.     

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.     

Overstimulation of PrPC signaling pathways by prion peptide 106-126 causes oxidative injury of bioaminergic neuronal cells

Transmissible spongiform encephalopathies, also called prion diseases, are characterized by neuronal loss linked to the accumulation of PrP(Sc), a pathologic variant of the cellular prion protein (PrP(C)). Although the molecular and cellular bases of PrP(Sc)-induced neuropathogenesis are not yet fully understood, increasing evidence supports the view that PrP(Sc) accumulation interferes with PrP(C) normal function(s) in neurons. In the present work, we exploit the properties of PrP-(106-126), a synthetic peptide encompassing residues 106-126 of PrP, to investigate into the mechanisms sustaining prion-associated neuronal damage. This peptide shares many physicochemical properties with PrP(Sc) and is neurotoxic in vitro and in vivo. We examined the impact of PrP-(106-126) exposure on 1C11 neuroepithelial cells, their neuronal progenies, and GT1-7 hypothalamic cells. This peptide triggers reactive oxygen species overflow, mitogen-activated protein kinase (ERK1/2), and SAPK (p38 and JNK1/2) sustained activation, and apoptotic signals in 1C11-derived serotonergic and noradrenergic neuronal cells, while having no effect on 1C11 precursor and GT1-7 cells. The neurotoxic action of PrP-(106-126) relies on cell surface expression of PrP(C), recruitment of a PrP(C)-Caveolin-Fyn signaling platform, and overstimulation of NADPH-oxidase activity. Altogether, these findings provide actual evidence that PrP-(106-126)-induced neuronal injury is caused by an amplification of PrP(C)-associated signaling responses, which notably promotes oxidative stress conditions. Distorsion of PrP(C) signaling in neuronal cells could hence represent a causal event in transmissible spongiform encephalopathy pathogenesis.     

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.     

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.     

Immunomodulation of the human prion peptide 106-126 aggregation

Site-directed monoclonal antibodies (mAbs) may interact with their antigens, leading to stabilization, refolding, and suppression of aggregation. In the following study, we show that mAbs raised against the peptide 106-126 of human prion protein (PrP 106-126) modulate the conformational changes occurring in the peptide exposed to aggregation conditions. MAbs 3-11 and 2-40 prevent PrP 106-126's fibrillar aggregation, disaggregates already formed aggregates, and inhibits the peptide's neurotoxic effect on the PC12 cells system, while mAb 3F4 has no protective effect. We suggest that there are key positions within the PrP 106-126 molecule where unfolding is initiated and their locking with specific antibodies may maintain the prion peptide native structure, reverse the aggregated peptide conformation, and lead to rearrangements involved in the essential feature of prion diseases.     

Topology determination and functional analysis of the Escherichia coli TatC protein

Misfolding of the prion protein yields amyloidogenic isoforms, and it shows exacerbating neuronal damage in neurodegenerative disorders including prion diseases. Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) potently stimulate neuritogenesis and survival of neuronal cells in the central nervous system. Here, we tested these neuropeptides on neurotoxicity in PC12 cells induced by the prion protein fragment 106-126 [PrP (106-126)]. Concomitant application of neuropeptide with PrP(106-126) (5x10(-5) M) inhibited the delayed death of neuron-like PC12 cells. In particular, PACAP27 inhibited the neurotoxicity of PrP(106-126) at low concentrations (>10(-15) M), characterized by the deactivation of PrP(106-126)-stimulated caspase-3. The neuroprotective effect of PACAP27 was antagonized by the selective PKA inhibitor, H89, or the MAP kinase inhibitor, U0126. These results suggest that PACAP27 attenuates PrP(106-126)-induced delayed neurotoxicity in PC12 cells by activating both PKA and MAP kinases mediated by PAC1 receptor.     

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.     

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.     

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.     

Interaction of the 106-126 prion peptide with lipid membranes and potential implication for neurotoxicity

Prion diseases are fatal neurodegenerative disorders characterized by the accumulation in the brain of an abnormally misfolded, protease-resistant, and beta-sheet rich pathogenic isoform (PrP(SC)) of the cellular prion protein (PrP(C)). In the present work, we were interested to study the mode of prion protein interaction with the membrane using the 106-126 peptide and small unilamellar lipid vesicles as model. As previously demonstrated, we showed by MTS assay that PrP 106-126 induces alterations in the human neuroblastoma SH-SY5Y cell line. We demonstrated for the first time by lipid-mixing assay and by the liposome vesicle leakage test that PrP 106-126, a non-tilted peptide, induces liposome fusion thus a potential cell membrane destabilization, as supported by membrane integrity assay (LDH). By circular dichroism (CD) analysis we showed that the fusogenic property of PrP 106-126 in the presence of liposome is associated with a predominantly beta-sheet structure. These data suggest that the fusogenic property associated with a predominant beta-sheet structure exhibited by the prion peptides contributes to the neurotoxicity of these peptides by destabilizing cellular membranes. The latter might be attached at the membrane surface in a parallel orientation as shown by molecular modeling.     

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.     

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.     

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.     

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.     

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.     

Comparison of mRNA expression patterns of class B scavenger receptors in BV2 microglia upon exposure to amyloidogenic fragments of beta-amyloid and prion proteins

The inflammatory responses in Alzheimer's disease and prion diseases are dominated by microglia activation. Scavenger receptors have been recently related to the innate immune activation of microglia initiated by endogenous ligands. In this study, we investigated mRNA expression patterns of B class scavenger receptors CD36 and scavenger receptor B1 (SR-B1) in BV2 microglia upon exposure to amyloid fibril Aβ(1-42) and PrP(106-126), respectively. CD36 and SR-B1 showed similar mRNA expression patterns following each treatment. PrP(106-126) induced a rapid increase of CD36 and SR-B1 mRNA levels in the treated microglia, whereas Aβ(1-42) induced a delayed but persistent increase in the mRNA expression of CD36 and SR-B1. These results suggest a possible involvement of CD36 and SR-B1 in microglial interaction with amyloidogenic fragments of beta-amyloid and prion proteins.