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. 2010 Nov 19;285(47):36542-50.
doi: 10.1074/jbc.M110.157263. Epub 2010 Sep 13.

Role of alpha7 nicotinic acetylcholine receptor in calcium signaling induced by prion protein interaction with stress-inducible protein 1

Flavio H Beraldo  1 , Camila P Arantes, Tiago G Santos, Nicolle G T Queiroz, Kirk Young, R Jane Rylett, Regina P Markus, Marco A M Prado, Vilma R Martins
Affiliations

Role of alpha7 nicotinic acetylcholine receptor in calcium signaling induced by prion protein interaction with stress-inducible protein 1

Flavio H Beraldo et al. J Biol Chem. .

Abstract

The prion protein (PrP(C)) is a conserved glycosylphosphatidylinositol-anchored cell surface protein expressed by neurons and other cells. Stress-inducible protein 1 (STI1) binds PrP(C) extracellularly, and this activated signaling complex promotes neuronal differentiation and neuroprotection via the extracellular signal-regulated kinase 1 and 2 (ERK1/2) and cAMP-dependent protein kinase 1 (PKA) pathways. However, the mechanism by which the PrP(C)-STI1 interaction transduces extracellular signals to the intracellular environment is unknown. We found that in hippocampal neurons, STI1-PrP(C) engagement induces an increase in intracellular Ca(2+) levels. This effect was not detected in PrP(C)-null neurons or wild-type neurons treated with an STI1 mutant unable to bind PrP(C). Using a best candidate approach to test for potential channels involved in Ca(2+) influx evoked by STI1-PrP(C), we found that α-bungarotoxin, a specific inhibitor for α7 nicotinic acetylcholine receptor (α7nAChR), was able to block PrP(C)-STI1-mediated signaling, neuroprotection, and neuritogenesis. Importantly, when α7nAChR was transfected into HEK 293 cells, it formed a functional complex with PrP(C) and allowed reconstitution of signaling by PrP(C)-STI1 interaction. These results indicate that STI1 can interact with the PrP(C)·α7nAChR complex to promote signaling and provide a novel potential target for modulation of the effects of prion protein in neurodegenerative diseases.

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Figures

FIGURE 1.
FIGURE 1.
STI1 interaction with PrPC promotes intracellular Ca2+ increase. A and B, Prnp+/+ (A) or Prnp0/0 (B) hippocampal neurons loaded with Fluo-3 AM were treated with STI1 (1 μm) in medium supplemented with (solid lines) or without (dashed line) Ca2+. THG-treated Prnp0/0 neurons show normal levels of intracellular Ca2+ stores. C, Prnp+/+ neurons were treated with an STI1 deletion mutant, STI1Δ230–245, lacking the PrPC binding site. D, relative intracellular Ca2+ levels in Prnp0/0 (white bars) and Prnp+/+ (black bars) neurons treated with STI1 or STI1Δ230–245 in the presence or absence of CaCl2 are indicated. The results represent the mean ± S.E. (error bars) of four independent experiments, and statistical significance was determined by one-way ANOVA and Newman-Keuls post test. *, p < 0.05 compared with controls.
FIGURE 2.
FIGURE 2.
PKA activation and ERK1/2 phosphorylation are dependent on PrPC-STI1-induced Ca2+ influx. PKA (A) and ERK1/2 (B) activation was induced by STI1 treatment in Prnp+/+ hippocampal neurons. Experiments were performed with or without CaCl2, as indicated. Forskolin-treated cells were used as positive controls for PKA activation. Relative levels of ERK1/2 activity represent the ratio between phosphorylated ERK1/2 (upper panel) and total ERK1/2 (lower panel) normalized to the untreated group. The results represent the mean ± S.E. (error bars) of four independent experiments, and statistical significance was determined by one-way ANOVA and Newman-Keuls post test. *, p < 0.05 compared with controls.
FIGURE 3.
FIGURE 3.
PrPC-STI1 interaction induces Ca2+ influx, PKA and ERK1/2 activation through α7nAChR. A and B, intracellular Ca2+ levels in Prnp+/+ hippocampal neurons were treated with STI1 (1 μm) in the presence (dashed line) or absence (solid line) of VGCC inhibitors (n = 3) (A) or in the presence (dashed line) or absence (solid line) of αBgt, a specific inhibitor of α7nAChR (n = 4) (B). C and D, PKA activity (n = 3) (C) and ERK1/2 (n = 3) phosphorylation (D) were measured in Prnp+/+ hippocampal neurons treated with STI1 in the presence of αBgt, as indicated. Forskolin was used as a positive control for PKA activation. Relative levels of ERK1/2 activity represent the ratio between phosphorylated ERK1/2 (upper panel) and total ERK1/2 (lower panel), normalized to the untreated group. The results were compared by one-way ANOVA and Newman-Keuls post test. *, p < 0.05 compared with controls.
FIGURE 4.
FIGURE 4.
PrPC interacts with α7nAChR. A, HEK 293 cells were transfected with expression vector lacking insert (vector) or vectors encoding FLAG-tagged α7nAChR (α7) or FLAG-tagged α7nAChR and RIC3 (α7/RIC3). Cells were incubated with Alexa Fluor 647 αBgt and analyzed by confocal microscopy. Phase contrast images indicate the field of cells (top panels), and green fluorescence indicates Alexa Fluor 647 αBgt-labeled cells (bottom panels). B, HEK 293 cells were transfected with expression vector lacking insert (Vector) or expression vectors encoding PrPC either alone (PrPC) or co-transfected with FLAG-tagged α7nAChR and RIC3 (α7/RIC3). Immunoprecipitation (IP) was performed with anti-FLAG antibodies, and proteins from total lysates (Input) or eluates (IP-FLAG) were immunoblotted (IB) with anti-PrPC (IB-PrPC) or anti-FLAG (IB-FLAG) antibodies.
FIGURE 5.
FIGURE 5.
Expression of α7nAChR rescues PrPC-STI1-mediated Ca2+ influx in HEK 293 cells. A–F, HEK 293 cells were treated with 2 μm STI1 alone (A–C and E), STI1 in the presence of PrPC antibodies 6H4 or 3F4 (D), or 2 μm STI1 deleted of the PrPC binding site, STI1Δ230–245 (F). Cells were transfected with empty vector (n = 5) (A), PrPC (n = 4) (B), α7nAChR (n = 4) (C), α7nAChR and RIC3 (D) and treated with STI1 alone (n = 8) or in the presence of 6H4 (n = 4) or 3F4 (n = 3), PrPC, α7nAChR, and RIC3 (n = 4) (E) or α7nAChR and RIC3 (n = 5) (F). Cells were treated with THG, when indicated, to test the level of intracellular Ca2+ stores. G, relative levels of intracellular Ca2+ in cells transfected and treated as indicated are shown. The results represent the mean ± S.E. (error bars) of independent experiments compared by one-way ANOVA and Newman-Keuls post test. *, p < 0.01 compared with controls.
FIGURE 6.
FIGURE 6.
Expression of α7nAChR in HEK 293 cells rescues PrPC-STI1-induced PKA activity and ERK1/2 phosphorylation. PKA activity (A) and ERK1/2 phosphorylation (B) in HEK 293 cells transfected with empty vector or co-transfected with expression vectors for α7nAChR and RIC3. Cells were treated with forskolin, STI1, or choline chloride as indicated. Relative levels of ERK1/2 activity represent the ratio between phosphorylated ERK1/2 and total ERK1/2 (upper panel) in cells treated with STI1 and normalized to cells transfected with empty vector. The results represent the mean ± S.E. (error bars) of three independent experiments compared by one-way ANOVA and Newman-Keuls post test. *, p < 0.01 compared with controls.
FIGURE 7.
FIGURE 7.
PrPC-STI1 interaction promotes neuroprotection and neuritogenesis through α7nAChR. A, Prnp+/+ hippocampal neurons were treated with STI1 (30 min) or STI1 and αBgt, followed by staurosporine, as indicated. Cells were immunolabeled with anti-activated caspase-3, which labels apoptotic cells. Data are represented as the percentage of activated caspase-3-positive cells. B and C, Prnp+/+ hippocampal neurons were treated with STI1 alone, or STI1 plus αBgt, as indicated, and neuritogenesis was measured as the percentage of cells with neurites or with neurites >30 μm, respectively. The results represent the mean ± S.E. (error bars) of three independent experiments, compared by one-way ANOVA and Newman-Keuls post test. *, p < 0.01 compared with controls.
FIGURE 8.
FIGURE 8.
Schematic model of signaling events mediated by PrPC-STI1 interaction. PrPC-STI1 interaction modulates α7nAChR, leading to Ca2+ influx and PKA and ERK1/2 activation. PKA and ERK1/2 activation promotes neuroprotection and neuritogenesis, respectively.
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References

    1. Westergard L., Christensen H. M., Harris D. A. (2007) Biochim. Biophys. Acta 1772, 629–644 - PMC - PubMed
    1. Linden R., Martins V. R., Prado M. A., Cammarota M., Izquierdo I., Brentani R. R. (2008) Physiol. Rev. 88, 673–728 - PubMed
    1. Linden R., Martins V. R, Prado M. A. (2009) Prion protein. UCSD-Nature Molecule Pages, 29July2009, doi:10.1038/mp.a003935.01 - DOI
    1. Nimmrich V., Ebert U. (2009) Rev. Neurosci. 20, 1–12 - PubMed
    1. Walsh D. M., Selkoe D. J. (2007) J. Neurochem. 101, 1172–1184 - PubMed

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