Disease-associated mutations in the prion protein impair laminin-induced process outgrowth and survival

Abstract

Prions, the agents of transmissible spongiform encephalopathies, require the expression of prion protein (PrP(C)) to propagate disease. PrP(C) is converted into an abnormal insoluble form, PrP(Sc), that gains neurotoxic activity. Conversely, clinical manifestations of prion disease may occur either before or in the absence of PrP(Sc) deposits, but the loss of normal PrP(C) function contribution for the etiology of these diseases is still debatable. Prion disease-associated mutations in PrP(C) represent one of the best models to understand the impact of PrP(C) loss-of-function. PrP(C) associates with various molecules and, in particular, the interaction of PrP(C) with laminin (Ln) modulates neuronal plasticity and memory formation. To assess the functional alterations associated with PrP(C) mutations, wild-type and mutated PrP(C) proteins were expressed in a neural cell line derived from a PrP(C)-null mouse. Treatment with the laminin γ1 chain peptide (Ln γ1), which mimics the Ln binding site for PrP(C), increased intracellular calcium in cells expressing wild-type PrP(C), whereas a significantly lower response was observed in cells expressing mutated PrP(C) molecules. The Ln γ1 did not promote process outgrowth or protect against staurosporine-induced cell death in cells expressing mutated PrP(C) molecules in contrast to cells expressing wild-type PrP(C). The co-expression of wild-type PrP(C) with mutated PrP(C) molecules was able to rescue the Ln protective effects, indicating the lack of negative dominance of PrP(C) mutated molecules. These results indicate that PrP(C) mutations impair process outgrowth and survival mediated by Ln γ1 peptide in neural cells, which may contribute to the pathogenesis of genetic prion diseases.

FIGURE 1.

Expression of wild-type and mutant forms of PrP^C in PrP^C-null neural cells. The PrP^C-null neuronal cell line CF10 was transfected with an expression vector encoding wild-type PrP^C (PrP3F4) or PrP^C mutants Leu-101, Leu-104, Val-116, Asn-177, Ile-179, and Lys-199 and maintained as a non-clonal population. PrP^C expression was detected using the anti-3F4 and R-phycoerythrin conjugated secondary antibodies. A, shown is flow cytometry of non-permeabilized cells expressing wild-type or PrP^C mutants. B, immunofluorescence for PrP^C in permeabilized cells using anti-3F4 antibody (red) and DAPI (blue) is shown. C, Western blotting of cell extracts using the anti-3F4 antibody is shown.

FIGURE 2.

PrP^C mutants show minor resistance to proteinase K digestion. Extracts from CF10 cells expressing wild-type (PrP3F4) or PrP^C mutants were treated or not with increasing concentrations of PK (0, 0.5, 1, and 4 μg/ml) for 30 min at 4 °C. The condition where cells were treated with PK had three times more total protein than the undigested condition. The remaining undigested PrP^C was detected by Western blotting (anti-PrP^C antibody). A, the relative levels of PrP^C represent the ratio between the levels of PrP^C after PK treatment and the total load of PrP^C (control). Relative values are represented as the mean ± S.E. (n = 3). B, representative Western blots contain 3 times more total protein in the PK digestion conditions than in non-digested ones. The asterisk on the left side of the panel is to shown the migration of the molecular marker of 30 kDa.

FIGURE 3.

mGluR1 and mGluR5 mediate intracellular calcium increase upon PrP^C-Ln γ1 peptide binding. A and B, CF10 cells expressing PrP^C wild-type (PrP3F4) and their PrP^C-null counterpart (CF10) were loaded with 10 μm Fluo3 AM and treated with the mGluR agonist DHPG (100 μm). C and D, shown is Ln γ1 peptide (37 μm) or thapsigargin (THG) (1 μm) in a medium supplemented with 2 mm CaCl₂. Cells were also treated with Ln γ1 peptide or DHPG in the presence of mGluR1 antagonist (LY367385, 50 μm) (E and F), in the presence mGluR5 antagonist (MPEP, 5 μm) (G and H), or in the presence of both 50 μm LY367385 and 5 μm MPEP (I and J). K, relative intracellular calcium levels are expressed as the mean ± S.E. n = 3. *, p < 0.05 versus control; **, p < 0.001 versus control; #, p < 0.001 versus PrP3F4 treated with Ln γ1 peptide; §, p < 0.001 versus CF10 treated with DHPG.

FIGURE 4.

Partial impairment in calcium signaling mediated by Ln γ1 peptide in CF10 expressing mutant PrP^C. CF10 cells transfected with wild-type PrP^C (PrP3F4) (A), non-transfected (CF10) (B), or transfected with the PrP^C mutants Lys-101 (C), Lys-104 (D), Val-116 (E), Asn-177 (F), Ile-179 (G), and Lys-199 (H) were loaded with 10 μm Fluo-3 AM and treated with Ln γ1 peptide (37 μm) and thapsigargin (THG) (1 μm) in medium supplemented with 2 mm CaCl₂. I, relative intracellular calcium levels are expressed as the mean ± S.E. n = 7; *, p < 0.05; **, p < 0.01. J, shown is intracellular calcium concentration in cells treated or not with DHPG. Calcium levels at the untreated condition (white bars) were considered = 1, and the values after DHPG treatment (gray bars) were relative to it. Values represent the mean ± S.E. n = 3.

FIGURE 5.

Process outgrowth mediated by the Ln γ1-peptide can be rescued after the reconstitution of PrP^C expression in CF10 cells. A, CF10 cell lines with and without transfections of wild-type PrP^C (PrP3F4) were cultured 48 h on coverslips pretreated with 10 μg/ml full-length laminin or 5 μg/ml poly-l-lysine (PL). Cells were fixed and labeled, and process outgrowth was quantified. B and C, PrP3F4- and non-transfected CF10 cells were treated with 10 μg/ml mitomycin C or vehicle for 3 h, washed, and plated on PL for 48 h to assess. The total cell number (proliferation) after 48 h (B) or cell death (using trypan blue) (C) after 3 h of mitomycin treatment. Values represent the mean ± S.E. (n = 3). *, p < 0.05 versus control. D, PrP3F4- and non-transfected CF10 cells pretreated with 10 μg/ml mitomycin C for 3 h were cultured on 5 μg/ml PL, PL plus laminin (2.5 or 5 μg/ml), or PL plus Ln γ1 peptide (Pep g1) (16.8 or 37 μm) for 48 h. Values represent the mean ± S.E. n = 3. *, p < 0.05 compared with the control PrP3F4 or CF10 in PL; **, p < 0.05 compared with the control PrP3F4 in PL.

FIGURE 6.

Process outgrowth induced by Ln γ1 is impaired in cells expressing PrP^C mutants. PrP3F4- and non-transfected CF10 cells as well as CF10 cells expressing the PrP^C mutants Leu-101, Leu-104, Val-116, Asn-177, Ile-179, and Lys-199 were treated with 10 μg/ml mitomycin C or vehicle for 3 h washed and plated on poly-l-lysine for 48 h. A, shown is total cell number (proliferation). *, p < 0.05 versus control. B, shown is the percentage of cell death (using trypan blue) measured after 3 h of mitomycin C treatment. *, p < 0.05 versus control. C, shown is the percentage of cell death (capase-3 activation) using staurosporine (STS) as the positive control after 48 h of mitomycin C treatment. *, p < 0.05 versus control; #, p < 0.05 PrP3F4 STS treatment versus all others STS treatment. D, cells were treated with 10 μg/ml mitomycin C for 3 h, washed, plated on poly-l-lysine, and treated with 37 μm Ln γ1 scrambled (SCR) peptide or 37 μm Ln γ1 peptide (γ1) for 48 h. The percentage of cells with process outgrowth was evaluated (*, p < 0.05 versus al others). E, shown are representative images of CF10 cells expressing wild-type (PrP3F4) and PrP^C mutants treated with mitomycin C followed by treatment with 37 μm Ln γ1 peptide. The arrows show the structures considered as typical process outgrowth. For all experiments values represent the mean ± S.E. of at least three independent experiments.

FIGURE 7.

Cell survival promoted by Ln γ1 is impaired in cells expressing PrP^C mutants. PrP3F4- and non-transfected CF10 cells as well as CF10 cells expressing PrP^C mutants Leu-101, Leu-104, Val-116, Asn-177, Ile-179, and Lys-199 were plated on 5 μg/ml poly-l-lysine plus 37 μm Ln γ1 peptide (γ1) or 37 μm Ln γ1 SCR peptide and treated or not with 100 nm staurosporine (STS). A, quantification of active caspase 3-positive cells is shown. *, p < 0.01 Ln γ1 peptide plus STS treated CF10 and PrP^C mutants versus Ln γ1 peptide plus STS treated PrP3F4. B, PrP3F4, CF10 cells, and CF10 cells expressing the PrP^C mutants Lys-101 and Ile-179 were co-transfected with wild-type PrP^C (PrP3F4) and immunoblotted for PrP^C. C, cells were plated on 5 μg/ml poly-l-lysine plus 37 μm Ln γ1 peptide or 37 μm Ln γ1 SCR peptide and treated or not with 100 nm STS. Active caspase 3 positive cells were quantified. *, p < 0.01.