Cutting Edge: The Neurotoxic Prion Peptide Fragment PrP_106–126 Is a Chemotactic Agonist for the G Protein-Coupled Receptor Formyl Peptide Receptor-Like 1

Materials and Methods

Results and Discussion

Microglia, the resting monocytes in the brain, and peripheral blood monocytes accumulate and are activated at sites of prion plaques (2, 3). We therefore investigated the capacity of PrP_106–126 to induce directional migration of monocytes, a crucial step in the accumulation of cells at sites of origin of chemotactic factors. Freshly dissolved PrP_106–126 induced a dose-dependent migration of human monocytes with a maximal cell response occurring at 50 μM of the peptide (Figs. 1⇓ and 2⇓A), a concentration comparable to or lower than needed to activate other functions of microglia or monocytes (5, 7, 8, 9, 10). When incubated at 37°C (500 μM PrP_106–126 diluted in PBS) to promote the formation of fibrils, the aggregated peptide induced a comparable level of monocyte migration as freshly dissolved PrP_106–126 (Fig. 2⇓A). The monocyte migration is dependent on the chemotactic rather than the chemokinetic activity of the PrP_106–126, as evaluated with checkerboard analyses (data not shown). A peptide with a scrambled amino acid sequence of PrP_106–126 was completely inactive (data not shown). Since many known leukocyte chemotactic factors use G protein-coupled receptors to induce directional cell migration, we investigated whether PrP_106–126 also activated such a receptor. Monocyte migration in response to soluble PrP_106–126 was completely inhibited by treatment of the cells with PT, an inhibitor of G_i and G_o proteins (Fig. 2⇓B), suggesting that a G protein-coupled receptor was involved. This was supported by induction of transient mobilization of intracellular calcium (Ca²⁺) in monocytes by PrP_106–126 (Fig. 2⇓C), which was also completely inhibited by pretreatment of the cells with PT (data not shown).

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FIGURE 1.

Monocyte chemotaxis induced by PrP_106–126. Left, Monocyte migration in the absence of stimuli. Open arrow indicates a micropore on the polycarbonate filter. Filled arrow denotes a monocyte migrated across the filter. Center, Monocyte migration in response to 50 μM freshly dissolved PrP_106–126. Right, Monocyte migration induced by 10 nM bacterial chemotactic peptide fMLF. Original magnification, ×400.

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FIGURE 2.

Activation of human monocytes by PrP_106–126. A, Migration of monocytes induced by freshly prepared PrP_106–126 and PrP_106–126 “aged” at 37°C for 3 or 7 days. B, Effect of preincubation with medium (PT−) or PT+ (100 ng/ml, 37°C, 30 min) on monocyte migration to PrP_106–126. fMLF at 100 nM was used as a control. C, Ca²⁺ mobilization induced by PrP_106–126 in monocytes. D, Attenuation of PrP_106–126-induced Ca²⁺ flux in monocytes by fMLF.

We then examined the capacity of a variety of other chemoattractants to cross-desensitize PrP_106–126-induced Ca²⁺ mobilization. This method has been effectively used previously to identify the sharing of receptors by chemotactic factors. The bacterial chemotactic peptide fMLF clearly attenuated PrP_106–126-induced Ca²⁺ flux in monocytes (Fig. 2⇑D). Since high concentrations of fMLF were required to completely desensitize the cell response to PrP_106–126 (Fig. 2⇑D), and such concentrations of fMLF have been found to additionally activate a G protein-coupled receptor FPRL1, which is also termed as the low-affinity fMLF receptor based on its homology to the high-affinity fMLF receptor FPR (13), we postulated that PrP_106–126 might share FPRL1 with fMLF. It was therefore determined that PrP_106–126 induced significant Ca²⁺ mobilization in FPRL1/293 cells (Fig. 3⇓A), which was also completely inhibited by PT (data not shown). Untransfected parental cells (data not shown) or cells transfected with other chemoattractant receptors including FPR (Fig. 3⇓E) did not respond to PrP_106–126. PrP_106–126 signaling in FPRL1/293 cells was desensitized by prior stimulation of the cells with high concentrations of fMLF (data not shown) and bidirectionally by another ligand for FPRL1, a synthetic peptide MMK-1 (14) (Fig. 3⇓, B–D). Furthermore, FPRL1/293 cells, but not parental 293 cells or cells transfected with FPR, migrated in response to PrP_106–126 (Fig. 3⇓F). The concentrations required for PrP_106–126 to activate FPRL1 were similar to those for monocytes, suggesting a major role for FPRL1 in monocyte activation by this prion protein fragment.

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FIGURE 3.

Activation of FPRL1/293 cells by PrP_106–126. A, Ca²⁺ mobilization induced by PrP_106–126 in FPRL1/293 cells. B–D, Cross-desensitization of calcium mobilization between PrP_106–126 and MMK-1 in FPRL1/293 cells. E, Ca²⁺ flux induced by fMLF and PrP_106–126 in FPR-expressing RBL cells (ETFR). F, Migration of FPRL1/293 and ETFR cells induced by PrP_106–126. fMLF at 100 nM was used as a control. G, Confocal micrographs of FPRL1/293 cells stained with anti-FPRL1 Ab (green) showing localization of FPRL1 in the cell membrane (left). FPRL1 internalization is induced by PrP_106–126 (50 μM; center) and WKYMVm (W peptide, 2 μM; right) in these cells. The nuclei appear blue (DAPI counterstain). Original magnification, ×600.

The possibility that FPRL1 contributed to the intracellular accumulation of prion fibrils was examined. Confocal microscopy showed that PrP_106–126 induced a rapid internalization of FPRL1 in FPRL1/293 cells (Fig. 3⇑G). As a peptide control, it was shown that another synthetic peptide WKYMVm (W peptide), a ligand for FPRL1 (12), similarly induced internalization of FPRL1. These results suggest that FPRL1 may also play a role in the uptake of prion peptide by FPRL1-bearing cells.

We further investigated the capacity of ligand activation of FPRL1 to enhance the production of proinflammatory cytokines in monocytes. PrP_106–126 at concentrations that induced FPRL1-mediated cell migration stimulated a PT-sensitive production of proinflammatory cytokines in monocytes (Table I⇓). The FPRL1 ligand MMK-1 had similar effects. In contrast, cytokine production in response to bacterial LPS, which does not use G protein-coupled receptor, was resistant to PT. Thus, in addition to mediating cell migration, FPRL1 activation by PrP_106–126 induced the release of proinflammatory cytokines that have been implicated in the neurotoxic effect of monocyte supernatants (10).

PrP_106–126 was derived from the amino acid sequence of human prion. In vitro, this peptide fragment has a high propensity to form amyloid-like aggregates and to induce apoptotic death of neuronal cells (4, 5, 6, 10). It was subsequently established that the neurotoxic effect of PrP_106–126 requires the presence of microglia or monocytes (4, 5, 6, 10), and PrP_106–126 induces signaling events in these cells that typically involved activation of cell surface receptor(s) (7, 8, 9, 10). Our study identifies FPRL1 as a functional receptor used by PrP_106–126 to induce monocytic cell migration and activation.

FPRL1 is a G protein-coupled receptor that is capable of interacting with a number of agonists, including peptide domains derived from HIV-1 envelope proteins (13) and at least four host-derived molecules, serum amyloid A (15), lipid metabolite lipoxin A4 (16), β amyloid peptide,⁵, and cathelicidin (17). FPRL1 mediates the chemotactic activity of peptide agonists and serum amyloid A, which is an acute phase protein and forms amyloid deposit during chronic inflammation (18). Consequently, FPRL1 behaves as a “pattern recognition” receptor that is activated to transduce signals by a wide variety of unrelated ligands. Stimulation of FPRL1 by its chemotactic agonists triggers a series of G protein-mediated signaling cascades leading to cell adhesion, migration, protein tyrosine phosphorylation, release of reactive oxygen intermediates, as well as gene activation and production of proinflammatory cytokines (13). These properties of FPRL1 account for various biological activities reported for PrP_106–126. FPRL1 is expressed in a number of cell types including cells of the nonhematopoietic origin such as epithelia (13). We found that human astrocytoma cells express FPRL1 and can be activated by FPRL1-specific agonists (19). We also detected FPRL1 gene expression in a neuroblastoma cell line and in a murine microglial cell line N9 (Y. Le, unpublished observation). Whether normal human neurons and astroglial cells express the functional FPRL1 and its role in glial activation and neuronal destruction are under further investigation.

A relatively high concentration (50 μM) of PrP_106–126 was required to induce an FPRL1-dependent maximal monocyte chemotactic response. Therefore, PrP_106–126 appears to interact with FPRL1 with a relatively low affinity. However, many chemoattractants, including some chemokines, also show low-affinity interaction with their receptors and such interactions nevertheless contribute to the recruitment of leukocytes to the sites of inflammation (20). Although our results are based on in vitro models, they have repeatedly been shown to correlate directly with in vivo disease states (2, 3) in which infiltration of mononuclear phagocytes (monocytes and microglia) was found in and around prion disease lesions in association with proinflammatory reactions (2, 3). Thus, the low-affinity PrP_106–126 and FPRL1 interaction may help direct monocytes/microglia migrate to the vicinity of prion lesions which contain high concentrations of these amyloidogenic precursors and aggregated fragments including PrP_106–126 (4, 21, 22, 23, 24). Interestingly, a recent study revealed that PrP_106–126 was detected in brain lesions of some Alzheimer’s disease patients, suggesting the coexistence of prion disease pathology in Alzheimer’s disease (25). Our observation of FPRL1 as a functional receptor, used by PrP_106–126 to chemoattract and activate mononuclear phagocytes, should promote further assessment of this receptor as a mediator of proinflammatory responses in neurodegenerative diseases and as a potential therapeutic target.