Activation of Src family kinase ameliorates secretory trafficking in mutant prion protein cells

Abstract

Fatal familial insomnia (FFI), genetic Creutzfeldt-Jakob disease (gCJD), and Gerstmann-Sträussler-Scheinker (GSS) syndrome are neurodegenerative disorders linked to prion protein (PrP) mutations. The pathogenic mechanisms are not known, but increasing evidence points to mutant PrP misfolding and retention in the secretory pathway. We previously found that the D178N/M129 mutation associated with FFI accumulates in the Golgi of neuronal cells, impairing post-Golgi trafficking. In this study we further characterized the trafficking defect induced by the FFI mutation and tested the 178N/V129 variant linked to gCJD and a nine-octapeptide repeat insertion associated with GSS. We used transfected HeLa cells, embryonic fibroblasts and primary neurons from transgenic mice, and fibroblasts from carriers of the FFI mutation. In all these cell types, the mutant PrPs showed abnormal intracellular localizations, accumulating in the endoplasmic reticulum (ER) and Golgi. To test the efficiency of the membrane trafficking system, we monitored the intracellular transport of the temperature-sensitive vesicular stomatite virus glycoprotein (VSV-G), a well-established cargo reporter, and of endogenous procollagen I (PC-I). We observed marked alterations in secretory trafficking, with VSV-G accumulating mainly in the Golgi complex and PC-I in the ER and Golgi. A redacted version of mutant PrP with reduced propensity to misfold did not impair VSV-G trafficking, nor did artificial ER or Golgi retention of wild-type PrP; this indicates that both misfolding and intracellular retention were required to induce the transport defect. Pharmacological activation of Src family kinase (SFK) improved intracellular transport, suggesting that mutant PrP impairs secretory trafficking through corruption of SFK-mediated signaling.

Keywords: Src family kinase; genetic prion disease; prion protein; protein misfolding; protein trafficking.

Figure 1

Mutant PrPs localize abnormally in HeLa cells. HeLa cells were transfected with plasmids encoding WT, PG14, CJD, or FFI PrP-EGFP fusion protein. After 48 h cells were fixed, permeabilized, stained with anti-PDI (A) or anti-GM130 (B) polyclonal antibody followed by Alexa Fluor 647 (red)-conjugated anti-rabbit IgG secondary antibody, and reacted with Hoechst 33258 (blue) to stain the nuclei. Scale bar 10 μm. C, the number of cells showing PrP-EGFP on the plasma membrane (PM) with no or little colocalization with PDI or GM130/giantin, or intense colocalization with PDI (ER) or GM130/giantin (Golgi) was counted and expressed as a percentage of the total. Each bar indicates the mean ± SD of three independent experiments evaluating a total of 210 WT, 198 PG14, 191 CJD, and 204 FFI PrP-EGFP transfected cells. ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 versus WT by two-way ANOVA, Tukey’s post-hoc test.

Figure 2

VSV-G accumulates in the Golgi of HeLa cells expressing mutant PrPs.A, HeLa cells were transfected with plasmids encoding WT, PG14, CJD or FFI PrP, and VSV-G-EGFP fusion protein. After 24 h at 35 °C cells were fixed, permeabilized, stained with polyclonal anti-GM130 and monoclonal anti-PrP 12B2 antibodies followed by Alexa Fluor-conjugated anti-IgG secondary antibodies. Cells were viewed with green excitation/emission settings to detect VSV-G and blue excitation/emission settings to detect GM130. Scale bar 10 μm. B, percentages of cells accumulating VSV-G in the Golgi. Data are the mean ± SD of three independent experiments. ∗∗p < 0.01, ∗∗∗p < 0.001 versus vector, and ^#p < 0.05, ^##p < 0.01 versus WT by one-way ANOVA, Tukey’s post-hoc test. C, the normalized fluorescent density of VSV-G in the Golgi was measured in PrP-expressing cells and expressed as fold change over vector-transfected cells. Each bar indicates the mean ± SD of 65 to 80 cells from three independent experiments; ∗∗∗∗p < 0.0001 versus vector and WT by one-way ANOVA, Tukey’s post-hoc test.

Figure 3

Mutant PrP misfolding and aggregation are necessary to impair VSV-G transport.A, HeLa cells were transfected with plasmids encoding WT, M128V, PG14, or PG14ΔHC PrP. After 24 h at 35 °C cells were fixed, permeabilized, stained with polyclonal anti-GM130 and monoclonal anti-PrP 12B2 antibodies followed by Alexa Fluor-conjugated anti-IgG secondary antibodies. Cells were viewed with green excitation/emission settings to detect VSV-G and blue excitation/emission settings to detect GM130. Scale bar 10 μm. B, the normalized fluorescent density of VSV-G in the Golgi was measured and expressed as fold change over vector-transfected cells. Data are the mean ± SD of 20 to 40 cells. ∗∗∗∗p < 0.0001 versus vector by one-way ANOVA, Tukey’s post-hoc test.

Figure 4

Retention of nonaggregated PrP in the ER or Golgi does not impair VSV-G trafficking.A, HeLa cells were transfected with plasmids encoding WT, PrP-ER, PrP-Golgi, or PG14 PrP. After 24 h cells were lysed in nondenaturing detergents, and the cell lysates were ultracentrifuged at 186,000g for 45 min. PrP in the supernatant (S) and pellet (P) was analyzed by western blot. Molecular weight markers are in kDa. B, HeLa cells were transfected with plasmids encoding WT, PrP-ER, PrP-Golgi or PG14 PrP, and VSV-G-EGFP fusion protein. After 24 h at 35 °C cells were fixed, permeabilized, stained with monoclonal anti-PrP 12B2 antibody followed by Alexa Fluor-conjugated anti-IgG secondary antibody. Cells were viewed with green excitation/emission settings to detect VSV-G and redexcitation/emission settings to detect PrP. Scale bar 10 μm. C, the normalized fluorescent density of VSV-G at the plasma membrane (PM) was measured in PrP-expressing cells and expressed as the fold difference from WT PrP-transfected cells. Each bar indicates the mean ± SD of 9 to 21 cells; ∗∗∗∗p < 0.0001 versus WT by one-way ANOVA, Tukey’s post-hoc test.

Figure 5

VSV-G transport is impaired in primary cells from mutant PrP but not PrP knockout mice.A, MEFs from WT, CJD, FFI, and PrP knockout (KO) mice were infected with VSV at 32 °C for 1 h, washed, and cultured at 35 °C for 4 h. Cells were fixed and immunostained with monoclonal anti-VSV-G and polyclonal anti-GM130 antibodies, followed by Alexa Fluor-conjugated anti-IgG secondary antibodies. Cells were viewed with green excitation/emission settings to detect VSV-G and red excitation/emission settings to detect GM130. Scale bar 50 μm. B, the normalized fluorescent density of VSV-G in the Golgi was measured and expressed as fold change over WT. Each bar indicates the mean ± SD of 22 to 37 cells; ∗∗∗∗p < 0.0001 versus WT and KO by one-way ANOVA, Tukey’s post-hoc test. C, primary hippocampal neurons from WT, CJD, and FFI mice were infected with VSV at 32 °C for 45 min, washed and cultured at 35 °C for 2 h. Cells were fixed and immunostained with monoclonal anti-VSV-G and polyclonal anti-giantin antibodies, followed by Alexa Fluor-conjugated anti-IgG secondary antibodies. Cells were viewed with green excitation/emission settings to detect VSV-G and red excitation/emission settings to detect giantin. Scale bar 10 μm.

Figure 6

Abnormal intracellular PrP localization and impairment of VSV-G transport in human fibroblasts from carriers of the FFI mutation.A, to visualize PrP on the cell surface, human fibroblasts (HFs) from noncarriers (WT) and carriers of the PRNP D178N mutation (FFI) were immunostained with anti-PrP antibody 6H4 before fixation and application of Alexa Fluor 488 (green)-conjugated secondary antibody and staining with Hoechst 33258 (blue) to visualize the nuclei. To investigate the intracellular distribution of PrP, cells were fixed and permeabilized before immunostaining. B, FFI HFs were fixed, permeabilized, and immunostained with anti-PrP and anti-giantin antibodies, followed by Alexa Fluor-conjugated secondary antibodies. Cells were viewed with green excitation/emission settings to detect PrP, red excitation/emission settings to detect giantin, and UV excitation/emission settings to detect the nuclei. Scale bar 15 μm. C, HFs were infected with VSV at 32 °C for 1 h, washed, and cultured at 35 °C for 4 h. Cells were fixed and immunostained with monoclonal anti-VSV-G and polyclonal anti-GM130 antibodies, followed by Alexa Fluor-conjugated IgG secondary antibodies. Scale bar 10 μm. D, the normalized fluorescent density of VSV-G in the Golgi was measured and expressed as fold change over WT. Each bar indicates the mean ± SEM of 209 WT and 211 FFI cells (four independent lines each). ∗∗∗∗p < 0.0001 by unpaired t-test. Scale bar 10 μm.

Figure 7

Electron microscopy localization of VSV-G in wild-type and FFI human fibroblasts. Cultures of fibroblasts from non-carriers (WT; A) and carriers of the PRNP D178N mutation (FFI; B and C) were infected with VSV at 32 °C for 1 h, washed, and cultured at 35 °C for 4 h. Cells were fixed and labeled with anti-VSV-G monoclonal antibody using the gold-enhance protocol. Scale bars 250 nm in A and B; 500 nm in C. D, quantification of gold particles in different cell compartments. Bars indicate the mean ± SD of 20 to 37 images. ∗∗∗∗p < 0.0001 versus WT by two-way ANOVA Bonferroni’s post-hoc test.

Figure 8

Ultrastructural abnormalities of the ER and Golgi complex in a FFI fibroblast.A, electron micrograph of a FFI fibroblast. B, three-dimensional tomography reconstruction from virtual serial slices of the fibroblast shown in A. The dilated ER cisterna is colored green. The continuous distensions of trans-Golgi are in pale yellow/dark red, the cis-most cisterna of Golgi stack is light blue. COP I and clathrin-coated (red arrow) vesicles are indicated. Scale bars 200 nm.

Figure 9

Src activation partially rescues the trafficking impairment of procollagen-I in FFI fibroblasts.A, cultures of human fibroblasts from noncarriers (WT) and carriers of the PRNP D178N mutation (FFI) were fixed, permeabilized, and immunostained with anti-PC-I and anti-GM130 antibodies. After incubation with Alexa Fluor-conjugated IgG secondary antibodies, cells were viewed with red excitation/emission settings to detect PC-I and green excitation/emission settings to detect GM130. B, HFs were treated with 10 μM Src Family activator or vehicle. After 4 h cells were fixed and stained with anti-PC-I antibody (red) and reacted with Hoechst 33258 (blue) to stain the nuclei. The bar graph indicates the percentages of cells with intracellular accumulation of PC-I. Data are mean ± SD of ∼2000 cells from four separate experiments; ∗∗∗p < 0.001 versus WT vehicle; ^#p < 0.05 versus FFI vehicle by two-way ANOVA, Tukey’s post-hoc test. Scale bars 30 μm.

Figure 10

Electron microscopy localization of procollagen-I in fibroblasts of carriers of the FFI mutation.A and B, immunogold staining on cryosections from FFI human fibroblasts using anti-PC-I, showing PC-I accumulation in dilated ER cisternae and in the Golgi. C, double immunogold staining using anti-PC-I (5 nm gold particles) and anti-GM130 antibodies (10 nm gold particles), showing accumulation of PC-I in the trans- but not in the cis- (GM130-positive) cisternae of the Golgi complex. Scale bars 500 nm. D, quantification of gold particles in different cell compartments. Bars indicate the mean ± SD of 5 to 26 images. ∗p < 0.05; ∗∗∗∗p < 0.0001 versus WT by two-way ANOVA Bonferroni’s post-hoc test.