A cationic tetrapyrrole inhibits toxic activities of the cellular prion protein

Abstract

Prion diseases are rare neurodegenerative conditions associated with the conformational conversion of the cellular prion protein (PrP(C)) into PrP(Sc), a self-replicating isoform (prion) that accumulates in the central nervous system of affected individuals. The structure of PrP(Sc) is poorly defined, and likely to be heterogeneous, as suggested by the existence of different prion strains. The latter represents a relevant problem for therapy in prion diseases, as some potent anti-prion compounds have shown strain-specificity. Designing therapeutics that target PrP(C) may provide an opportunity to overcome these problems. PrP(C) ligands may theoretically inhibit the replication of multiple prion strains, by acting on the common substrate of any prion replication reaction. Here, we characterized the properties of a cationic tetrapyrrole [Fe(III)-TMPyP], which was previously shown to bind PrP(C), and inhibit the replication of a mouse prion strain. We report that the compound is active against multiple prion strains in vitro and in cells. Interestingly, we also find that Fe(III)-TMPyP inhibits several PrP(C)-related toxic activities, including the channel-forming ability of a PrP mutant, and the PrP(C)-dependent synaptotoxicity of amyloid-β (Aβ) oligomers, which are associated with Alzheimer's Disease. These results demonstrate that molecules binding to PrP(C) may produce a dual effect of blocking prion replication and inhibiting PrP(C)-mediated toxicity.

Figure 1. Fe(III)-TMPyP binds to PrP^C.

(A) Structure of Fe(III)-TMPyP; (B) Fe(III)-TMPyP but not GN8, binds to recombinant PrP^C, as assayed by equilibrium dialysis. A 50 μM aliquot of the indicated compounds and recombinant proteins were placed in the assay and sample chambers, respectively. The chambers were separated by a 5,000 molecular weight cut-off membrane. Samples were left to equilibrate at room temperature for 1 day with gentle rocking. The concentration of each compound was quantified using UV-visible spectroscopy with buffer background subtracted. Statistics (by student t-test) was as follow: for GN8, Buffer vs BSA, p = 0.233; Buffer vs PrP^C, p = 0.394; for Fe(III)-TMPyP, Buffer vs BSA, p = 0.129; Buffer vs PrP^C, p = 3.9 × 10⁻⁵ (*). (C) Fe(III)-TMPyP binds to recombinant PrP^C, as detected by DMR. Different concentrations of Fe(III)-TMPyP were added to label-free microplate well surfaces (EnSpire-LFB HS microplate, Perkin Elmer) on which full-length mouse recombinant PrP^C had previously been immobilized. Measurements were performed before (baseline) and after (final) adding the compound. The response (pm) was obtained subtracting the baseline output to the final output signals. The output signal for each well was obtained by subtracting the signal of the protein-coated reference area to the signal of uncoated area. The data (red dots) were fitted (black line) to a sigmoidal function using a 4 parameter logistic (4PL) nonlinear regression model; R² = 0.96; p = 4.4 × 10⁻³. (D) SPR analyses of Fe(III)-TMPyP interaction with PrP^C. Starting at time 0, the indicated concentrations of Fe(III)-TMPyP were injected for 200 sec over sensor chip surfaces (GL-H chip, Bio-Rad) on which 16.000 resonance units (RU) of full-length, mouse recombinant PrP^C had previously been captured by amine coupling. The chip was then washed with PBST buffer alone to monitor ligand dissociation. Sensorgrams show Fe(III)-TMPyP binding in RU. The data were obtained by subtracting the reference channels, and best fitted by the Langmuir equation, assuming a heterogeneous ligand on the surface. Kinetic constants were as follow: k_a = 5.95 × 10² 1/Ms; k_d = 7.18 × 10⁻⁴ 1/s; K_d = 1.21 μM; R_max = 500.57 RU.

Figure 2. Fe(III)-TMPyP inhibits the replication of 22L prions in cell cultures.

22L-infected N2a cells were incubated with various concentrations (indicated) of Fe(III)-TMPyP for 72 h. The total amount of PrP^Sc was estimated in cell lysates by detecting the amount of protease K (PK)-resistant PrP by Western blot (upper panel), and quantifying signals by densitometric analysis (graph in the lower panel). Total PrP signal was revealed with anti-PrP^C antibody 6D11. Statistically-significant differences (*) between Fe(III)-TMPyP-treated and the untreated samples was estimated by student t-test: [1 μM], p = 0.034; [3 μM], p = 6.1 × 10⁻⁶; [10 μM], p = 3.4 × 10⁻⁷.

Figure 3. Fe(III)-TMPyP inhibits in vitro replication of vole-adapted scrapie.

(A) Tg338 brain homogenates, seeded with indicated dilutions of Dawson isolate scrapie strain, were subjected to a single, 48 hr-long round of standard PMCA, upon incubation with Fe(III)-TMPyP (500 μM) or vehicle (VHC) control. Amplified samples were digested with PK and analyzed by Western blot using monoclonal antibody SAF83. Fe(III)-TMPyP showed an inhibitory activity toward prion amplification of at least 16,000 folds. CTR: Normal, unseeded and untreated brain homogenate. Molecular markers are KDa. (B) Brain homogenates from terminally ill voles infected with an Italian vole-adapted scrapie strain were diluted 1:10 (F, lanes 1 and 3) or 1:100 (A, lanes 2 and 4) in PMCA substrate in presence of vehicle (VHC, lanes 1–2) or 500 μM Fe(III)-TMPyP (lanes 3 and 4). Samples diluted 1:100 were subjected to a single PMCA round, while those diluted 1:10 were kept frozen and used to determine the amplification factor. Samples were PK-digested and analyzed by Western Blotting with antibody SAF84. (C) A similar experiment was carried out, in triplicate (n = 3), using different concentrations of Fe(III)-TMPyP (1–100 μM). The graph illustrates mean amplification factors (±standard error) obtained with increasing concentrations of Fe(III)-TMPyP or vehicle alone. Statistical differences (*) between Fe(III)-TMPyP and vehicle control were estimated by student t-test: [1 μM], p = 0,476; [5 μM], p = 0,273; [10 μM], p = 0,073; [50 μM], p = 0,047; [100 μM], p = 0,019.

Figure 4. Fe(III)-TMPyP inhibits ionic currents induced by ΔCR PrP.

(A) Whole‐cell patch clamp recording from HEK293 cells expressing either WT or ΔCR PrP, at a holding potential of −80 mV. Fe(III)-TMPyP at the reported concentrations was added to the dish at the time indicated by the arrows. (B) Inward currents recorded from WT or ΔCR PrP HEK293 cells were plotted as the percentage of total time the cells exhibited currents ≥450 pA (mean ± S.E.M., n ≥ 5 cells), at a holding potential of −80 mV. Statistically-significant differences (*) between Fe(III)-TMPyP-treated and the untreated ΔCR cells were estimated by student t-test: [1], p = 0.036; [3], p = 0.0016; [10], p = 0.0014; [50], p = 0.005.

Figure 5. Fe(III)-TMPyP inhibits the drug-hypersensitizing effect of ΔCR PrP.

(A) The DBCA was used to evaluate the anti-ΔCR PrP effects of Fe(III)-TMPyP. Stably transfected WT or ΔCR HEK293 cells carrying the hygromycin B resistance cassette were plated in 24-well plates and incubated in medium containing 500 μg/mL of Zeocin, for 48 h at 37 °C. The picture shows an example of treated vs untreated wells after MTT assay. (B) The bar graph shows a quantification of the dose-dependent, rescuing effect of Fe(III)-TMPyP. Average values were obtained from a minimum of 4 independent experiments (n = 4), and expressed as a percentage of cell viability in untreated cells. Statistically-significant differences (*) between Fe(III)-TMPyP-treated and untreated cells was estimated by student t-test: [0.1], p = 0.1582; [0.3], p = 0.0659; [1], p = 1.67 × 10⁻⁴; [3], p = 2.19 × 10⁻⁴; [10], p = 3.37 × 10⁻⁶. (C) Fe(III)-TMPyP did not rescue the toxicity of Zeocin in WT PrP-expressing cells. The DBCA was adapted to test the toxicity of Zeocin in HEK293 cells stably expressing WT PrP, and evaluate the potential rescuing effect of Fe(III)-TMPyP. Cells were plated in 24-well plates and incubated in medium containing different concentrations (0–2.000 μg/mL) of Zeocin, for 72 h at 37 °C, in presence (1–10 μM) or absence of Fe(III)-TMPyP. Average values were obtained from a minimum of 3 independent experiments (n = 3), and expressed as percentage of cell viability of Zeocin-untreated cells.

Figure 6. Treatment with Fe(III)-TMPyP does not alter PrP^C expression.

(A) HEK293 cells expressing ΔCR PrP were treated with Fe(III)-TMPyP at different concentrations, and for the indicated time points. Total PrP levels were evaluated in whole-cell lysates by western blot, using anti-PrP antibody 6D11. The picture in the upper panel illustrates a Western blot related to the 3 h timepoint. Graph in the bottom panel shows the quantification of PrP levels, obtained by densitometric analysis of the Western blots, normalizing each value on the corresponding Ponceau S-stained lane. Bars represent the mean of two (20 min) to four (±SEM) independent experiments, expressed as percentage of the levels in untreated cells. (B) Hippocampal neurons from C57BL/6 mice were exposed to 10 μM Fe(III)-TMPyP (+) or the vehicle (−) for 24 h. Cells were lysed and analyzed by Western blot with anti-PrP (6D11) or anti-actin antibodies.

Figure 7. Fe(III)-TMPyP inhibits Aβ oligomer-dependent activation of Fyn.

(A) Treatment with Aβ oligomers induced a rapid phosphorylation of the Fyn kinase, which was fully prevented by pre-incubation with Fe(III)-TMPyP. Primary hippocampal neurons were pre-treated for 20 minutes with Fe(III)-TMPyP (10 μM) or vehicle control (volume equivalent) prior to incubation with Aβ oligomers (3 μM, monomer equivalent concentration) for 20 minutes. Triton-insoluble fractions were analyzed by immunoblot with antibodies against phospho-SFK (Tyr 416) or total Fyn. Actin was used as loading control. The picture shows an example of a Western blot for p-Fyn and Fyn. The graph reports the quantification of 5 independent experiments (n = 5). Values are expressed as percentage of vehicle (VHC)-treated cells, normalized on the intensity of the corresponding Actin bands. *Statistical significance was estimated by one-way ANOVA, Tukey post-hoc test (p = 3.77 × 10⁻⁴). (B) Neither Aβ, nor Fe(III)-TMPyP altered the phosphorylation of Fyn in PrP-null neurons. Primary hippocampal neurons derived from PrP KO mice were pre-treated for 20 minutes with Fe(III)-TMPyP (10 μM) or DMSO (volume equivalent) prior to incubation with Aβ oligomers (3 μM, monomer equivalent concentration) or vehicle (VHC, volume equivalent) for 20 minutes. Triton-insoluble fractions were analyzed by immunoblot with antibodies against phospho-SFK (Tyr 416) or total Fyn. Actin was used as loading control. The upper panel shows an example of a Western blot for p-Fyn and total Fyn. The bar graph reports the quantification of four independent experiments (n = 4). Values are expressed as percentage of VHC-treated cells, normalized on the intensity of the corresponding actin bands.

Figure 8. Fe(III)-TMPyP rescues the synaptotoxic effects of Aβ oligomers.

Aβ oligomers induced a loss of post-synaptic markers, which was significantly attenuated by pre-incubation with Fe(III)-TMPyP. Primary hippocampal neurons were pre-treated for 20 min with or without Fe(III)-TMPyP (10 μM), and exposed for 3 h to Aβ oligomers (3 μM) or DMSO vehicle (VHC). Post-synaptic proteins from triton-insoluble fractions were then measured by Western blotting. The picture shows a representative Western blot for each synaptic marker. Quantitation of multiple experiments is shown in the graph. Actin levels were not significantly affected by Aβ oligomer treatment. *Statistical significance. P values, calculated by one-way ANOVA, Tukey post-hoc test, were as follow: GluN2A, p = 0.013542; GluN2B, p = 0.010834; GluA1, p = 0.002597; GluA2, p = 0.015558; PSD95, p = 0.036794.