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.     

PrP 106-126 Altered PrP mRNA gene expression in mouse microglia BV-2 Cells

Prion diseases are infectious and fatal neurodegenerative diseases. The pathogenic agent is an abnormal prion protein aggregate. Microglial activation in the centre nervous system is a characteristic feature of prion disease. In this study, we examined the effect of PrP 106–126 on PrP mRNA gene expression in Mouse microglia cells BV-2 by real-time quantitative PCR. PrP mRNA expression level was found to be significantly increased after 18 h exposure of BV-2 cells to PrP 106–126, with 3-fold increase after 18 h and 4.5-fold increase after 24 h and BV-2 cells proliferating occurred correspondingly. Our results provide the first in vitro evidence of the increase of PrP mRNA levels in microglial cells exposed to PrP 106–126, and indicate that microglial cells might play a critical role in prion pathogenesis.     

Prion Peptide Uptake in Microglial Cells – The Effect of Naturally Occurring Autoantibodies against Prion Protein

In prion disease, a profound microglial activation that precedes neurodegeneration has been observed in the CNS. It is still not fully elucidated whether microglial activation has beneficial effects in terms of prion clearance or whether microglial cells have a mainly detrimental function through the release of pro-inflammatory cytokines. To date, no disease-modifying therapy exists. Several immunization attempts have been performed as one therapeutic approach. Recently, naturally occurring autoantibodies against the prion protein (nAbs-PrP) have been detected. These autoantibodies are able to break down fibrils of the most commonly used mutant prion variant PrP106-126 A117V and prevent PrP106-126 A117V-induced toxicity in primary neurons. In this study, we examined the phagocytosis of the prion peptide PrP106-126 A117V by primary microglial cells and the effect of nAbs-PrP on microglia. nAbs-PrP considerably enhanced the uptake of PrP106-126 A117V without inducing an inflammatory response in microglial cells. PrP106-126 A117V uptake was at least partially mediated through scavenger receptors. Phagocytosis of PrP106-126 A117V with nAbs-PrP was inhibited by wortmannin, a potent phosphatidylinositol 3-kinase inhibitor, indicating a separate uptake mechanism for nAbs-PrP mediated phagocytosis. These data suggest the possible mechanisms of action of nAbs-PrP in prion disease.     

c-Abl Tyrosine Kinase Mediates Neurotoxic Prion Peptide-Induced Neuronal Apoptosis via Regulating Mitochondrial Homeostasis

Prion diseases are neurodegenerative disorders characterized by the accumulation of a disease-associated prion protein and apoptotic neuronal death. Previous studies indicated that the ubiquitous expression of c-Abl tyrosine kinase transduces a variety of extrinsic and intrinsic cellular signals. In this study, we demonstrated that a synthetic neurotoxic prion fragment (PrP106-126) activated c-Abl tyrosine kinase, which in turn triggered the upregulation of MST1 and BIM, suggesting the activation of the c-Abl-BIM signaling pathway. The peptide fragment was found to result in cell death via mitochondrial dysfunction in neuron cultures. Knockdown of c-Abl using small interfering RNA protected neuronal cells from PrP106-126-induced mitochondrial dysfunction, production of reactive oxygen species, and apoptotic events inducing translocation of Bax to the mitochondria, cytochrome c release into the cytosol, and activation of caspase-9 and caspase-3. Blocking the c-Abl tyrosine kinase also prevented neuronal cells from PrP106-126-induced apoptotic morphological changes. This is the first study reporting that c-Abl tyrosine kinase as a novel upstream activator of MST1 and BIM plays an important role in prion-induced neuron apoptosis via mitochondrial dysfunction. Our findings suggest that c-Abl tyrosine kinase is a potential therapeutic target for prion disease.     

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.     

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.     

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.     

Neurotoxic prion protein (PrP) fragment 106-126 requires the N-terminal half of the hydrophobic region of PrP in the PrP-deficient neuronal cell line

The cytotoxicity of aged PrP(106-126) was examined using an immortalized prion protein (PrP) gene-deficient neuronal cell line. The N-terminal half of the hydrophobic region (HR) but not the octapeptide repeat (OR) of PrP was required for aged PrP(106-126) neurotoxicity, suggesting that neurotoxic signals of aged PrP(106-126) are mediated by this region.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

PrP106-126多肽诱导凋亡细胞中14-3-3β的降解

本研究将PrP106-126多肽和HeLa细胞共孵育4h和8h,采用Hoechst染色分析发现PrP106-126诱导凋亡细胞细胞核出现不同程度的染色质浓集,固缩及碎裂的细胞凋亡征象。Western blotting检测发现PrP106-126诱导细胞中的多聚ADP核糖聚合酶(poly ADP-ribose polymerase,PARP)降解,提示PrP106-126通过caspase3途径引起细胞凋亡现象。PrP106-126诱导的细胞中14-3-3β在不同孵育时间也出现降解,而Real-time PCR检测14-3-3β mRNA未发生变化。本研究证明PrP106-126通过caspase3诱导HeLa细胞凋亡,并可导致抗凋亡蛋白14-3-3β的降解而加速凋亡的形成。