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. 2010 Jul 20;107(29):13147-52.
doi: 10.1073/pnas.1000784107. Epub 2010 Jul 6.

Prion protein interaction with stress-inducible protein 1 enhances neuronal protein synthesis via mTOR

Martín Roffé  1 , Flávio Henrique Beraldo, Romina Bester, Max Nunziante, Christian Bach, Gabriel Mancini, Sabine Gilch, Ina Vorberg, Beatriz A Castilho, Vilma Regina Martins, Glaucia Noeli Maroso Hajj
Affiliations

Prion protein interaction with stress-inducible protein 1 enhances neuronal protein synthesis via mTOR

Martín Roffé et al. Proc Natl Acad Sci U S A. .

Abstract

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases caused by the conversion of prion protein (PrP(C)) into an infectious isoform (PrP(Sc)). How this event leads to pathology is not fully understood. Here we demonstrate that protein synthesis in neurons is enhanced via PrP(C) interaction with stress-inducible protein 1 (STI1). We also show that neuroprotection and neuritogenesis mediated by PrP(C)-STI1 engagement are dependent upon the increased protein synthesis mediated by PI3K-mTOR signaling. Strikingly, the translational stimulation mediated by PrP(C)-STI1 binding is corrupted in neuronal cell lines persistently infected with PrP(Sc), as well as in primary cultured hippocampal neurons acutely exposed to PrP(Sc). Consistent with this, high levels of eukaryotic translation initiation factor 2alpha (eIF2alpha) phosphorylation were found in PrP(Sc)-infected cells and in neurons acutely exposed to PrP(Sc). These data indicate that modulation of protein synthesis is critical for PrP(C)-STI1 neurotrophic functions, and point to the impairment of this process during PrP(Sc) infection as a possible contributor to neurodegeneration.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
STI1–PrPC interaction enhances protein synthesis in a PI3K-mTOR and ERK1/2 dependent manner. (A) Prnp+/+ (open bars) or Prnp0/0 (filled bars) neurons were incubated with [35S]-methionine, followed by stimulation with STI1 or 100 ng/mL BDNF for 30 min. Graph shows percentage of increase of [35S]-methionine incorporation relative to control cells. (B) Polysome profiles from neurons without treatment (control, Left) or treated with 2.4 μM STI1 for 30 min (Right). (C) Neurons were incubated with 2.4 μM STI1, 80 μM PepSTI1230–245, 80μM PepSTI161–76, or 2.4 μM STI1Δ(Δ). Graph shows percentage change of [35S]-methionine incorporation relative to control cells. (D) Synaptosomes were treated with 2.4 μM STI1 for 30 min. Graph shows percentage of increase of [35S]-methionine incorporation relative to control. (E) Neurons were preincubated with Ly294002 (5 μM, Ly), rapamycin (20 nM, Rapa), PD98059 (50 μM, PD), or Actinomycin D (1.5 μM, ActD) for 15 min before addition of 2.4 μM STI1. Graph shows percentage of increase of [35S]-methionine incorporation relative to untreated cells. (A, C, and E) *P < 0.05, ANOVA followed by Tukey post hoc test. (D) *P < 0.05, Student t test.
Fig. 2.
Fig. 2.
STI1–PrPC interaction induces phosphorylation of Akt, p70S6K, 4E-BP2, and eIF4E. Prnp+/+ (open bars) or Prnp0/0 (filled bars) neurons were treated with 2.4 μM STI1 or STI1Δ(Δ) for the indicated times. Western blots were performed for (A) phospho-Akt and total-Akt, (B) phospho-p70S6K and actin, (C) phospho-4E-BP1 and actin, and (D) phospho-eIF4E and actin. All values are expressed relative to control. Where indicated, cells were preincubated for 1 h with Ly294002 (5 μM, Ly) or rapamycin (20 nM, Rapa). *P < 0.05, ANOVA followed by Tukey post hoc test.
Fig. 3.
Fig. 3.
PrPC–STI1–induced neuritogenesis and neuroprotection is dependent on PI3K and mTOR signaling. Neurons were cultured with 0.6 μM STI1 and Ly294002 (A and B) or STI1 and rapamycin (C and D) for 24 h. Morphometric quantification of the following parameters was performed: percentage of cells with neurites (A and C), percentage of cells with neurites longer than 30 μm (B and D). (E) Neurons were cultured with 1.2 μM STI1 and Ly294002 or rapamycin for 1 h, followed by addition of 25 nM staurosporine. After 24 h, cells were fixed and stained with propidium iodide. Graph shows the percentage of pyknotic cells. *P < 0.05, ANOVA followed by Tukey post hoc test.
Fig. 4.
Fig. 4.
Protein synthesis is partially impaired in PrPSc-infected cells. Mock (open bars) or 22L persistently infected (filled bars) N2a (A) or SN-56 (B) cells were preincubated with [35S]-methionine, followed by 2.4 μM STI1 or 5 μg/mL insulin stimulation for 30 min. Graph shows the percentage of [35S]-methionine incorporation relative to mock-infected cells. (C) Primary neurons exposed to mock (open bars) or 22L-infected (filled bars) brain extracts were preincubated with [35S]-methionine, followed by 2.4 μM STI1 or 5 μg/mL insulin for 30 min. Graph shows percentage of [35S]-methionine incorporation relative to untreated, mock-infected cells. *P < 0.05, ANOVA followed by Tukey post hoc test.
Fig. 5.
Fig. 5.
PrPSc infection leads to eIF2α phosphorylation. Mock-infected (open bars) or 22L-infected N2a (filled bars) were treated with 2.4 μM STI1 or 2.5 μg/mL tunicamycin (Tu) for 1 h. Western blots were performed for antiphosphorylated and total eIF2α (A), phospho-PKR and actin (B). Graphs show levels of phospho-eIF2α (A) or phospho-PKR (B) relative to control. (C) Mock-infected (open bars) or 22L–persistently infected SN-56 cells (filled bars) were subjected to Western blot with antiphosphorylated or total eIF2α. Graph shows levels of phospho-eIF2α relative to control. (D) Hippocampal neurons were exposed to mock (open bars) or 22L-infected brain extract (filled bars) and subjected to Western blot with antiphosphorylated or total eIF2α. Graph shows levels of phospho-eIF2α relative to control. (A) *P < 0.05, ANOVA followed by Tukey post hoc test. (B–D) *P < 0.05, Student t test.
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