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. 2013 Oct 29;110(44):E4160-9.
doi: 10.1073/pnas.1317164110. Epub 2013 Oct 15.

Drug Resistance Confounding Prion Therapeutics

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Free PMC article

Drug Resistance Confounding Prion Therapeutics

David B Berry et al. Proc Natl Acad Sci U S A. .
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Abstract

There is not a single pharmaceutical that halts or even slows any neurodegenerative disease. Mounting evidence shows that prions cause many neurodegenerative diseases, and arguably, scrapie and Creutzfeldt-Jakob disease prions represent the best therapeutic targets. We report here that the previously identified 2-aminothiazoles IND24 and IND81 doubled the survival times of scrapie-infected, wild-type mice. However, mice infected with Rocky Mountain Laboratory (RML) prions, a scrapie-derived strain, and treated with IND24 eventually exhibited neurological dysfunction and died. We serially passaged their brain homogenates in mice and cultured cells. We found that the prion strain isolated from IND24-treated mice, designated RML[IND24], emerged during a single passage in treated mice. Although RML prions infect both the N2a and CAD5 cell lines, RML[IND24] prions could only infect CAD5 cells. When passaged in CAD5 cells, the prions remained resistant to high concentrations of IND24. However, one passage of RML[IND24] prions in untreated mice restored susceptibility to IND24 in CAD5 cells. Although IND24 treatment extended the lives of mice propagating different prion strains, including RML, another scrapie-derived prion strain ME7, and chronic wasting disease, it was ineffective in slowing propagation of Creutzfeldt-Jakob disease prions in transgenic mice. Our studies demonstrate that prion strains can acquire resistance upon exposure to IND24 that is lost upon passage in mice in the absence of IND24. These data suggest that monotherapy can select for resistance, thus intermittent therapy with mixtures of antiprion compounds may be required to slow or stop neurodegeneration.

Keywords: antiprion therapeutics; bioluminescence imaging; drug discovery.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Prion strain nomenclature. Schematic of serial passages and treatment in mice, using the example of mice infected with the RML prion strain, treated with IND24 (green arrows) or vehicle (V; red arrows). Nomenclature for cells infected with each strain is denoted by appending the cell line name, as shown in gray.
Fig. 2.
Fig. 2.
Effects of IND24 and IND81 treatment on RML-infected Tg(Gfap-luc)/FVB mice. (A) Kaplan–Meier survival curves for infected mice treated with vehicle (red; n = 12), IND24 (solid green; n = 12) or IND81 (dashed green; n = 11). This color scheme also applies to BE and NO. (B) Mean brain bioluminescence signal for mice in A, measured approximately every week; error bars represent SEM. (C) Immunoblot of PK-resistant PrP from the brains of terminal RML-infected mice treated with vehicle (RML[V]), IND24 (RML[IND24]) or IND81 (RML[IND81]). PrPSc was detected with HuM-P Fab conjugated to HRP. IND81-treated samples were loaded at 15× the protein level of other samples. Molecular weight markers of migrated protein standards represent 30 and 20 kDa. (D) Average ratio of monoglycosylated to diglycosylated PrPSc in mice treated with vehicle (n = 7), IND24 (n = 7), or IND81 (n = 9), as indicated. Error bars represent SD. ***P < 0.001, n.s., not significant, Student’s t test. (E) Conformational stability of prions from IND24-treated (n = 6), IND81-treated (n = 6), and vehicle-treated (n = 6) RML-infected mice. Samples were exposed to increasing concentrations of GdnHCl (0–4 M, as indicated) then subjected to PK digestion. Points represent average percentage of PK-resistant PrP remaining at each concentration of GdnHCl, and error bars represent SD. (FM) Neuropathologic analysis of brain sections from RML[V] (Left) and RML[IND24] (Right) mice. (F and G) PrPSc immunohistochemistry of brainstem sections, stained using the R2 monoclonal antibody. (Scale bars in F and G: 100 μm.) (HM) H&E stain of tissue sections from the brainstem (H and I), lateral cortex (J and K), and cerebellum (L and M). (Scale bars in HM; 50 μm.) (N) Quantification of PrPSc deposition in multiple brain regions from RML-infected Tg(Gfap-luc)/FVB mice treated with IND24 (n = 12) or vehicle (n = 5), using the R2 monoclonal antibody. BF, basal forebrain; Bs, brainstem; CbG, cerebellum-granule cell layer; CbM, cerebellum-molecular cell layer; Cd, caudate nucleus; Hp, hippocampus; NC-L, neocortex-lateral; NC-M, neocortex-medial; Th, thalamus; Sp, septum. Error bars represent SEM. Measurements were made in intensity units (IU). (O) Quantification of vacuolation from multiple brain regions of mice treated with vehicle (n = 5) and IND24 (n = 14). Abbreviations as in N. Error bars represent SEM.
Fig. 3.
Fig. 3.
Infection of cultured cells with IND24-treated prions demonstrates IND24-resistance. PK-resistant PrPSc in N2a (A and B) and CAD5 (C and D) cells infected with RML[V] (A and C) and RML[IND24] (B and D) treated with 0–20 μM IND24, as indicated. PrPSc was assayed by immunoblot using HRP-conjugated HuM-D13 Fab. Images are representative of three replicates. Molecular weight markers of migrated protein standards represent 30 and 20 kDa. (E) Average ratio of monoglycosylated to diglycosylated PrPSc in CAD5 cells infected with RML[V] (n = 6) or RML[IND24] (n = 6). Error bars represent SD. ***P < 0.001, Student’s t test.
Fig. 4.
Fig. 4.
Serial passage of prions from IND24-treated mice. (A) Kaplan–Meier survival curves for passage of RML[IND24] and RML[IND24;IND24] into Tg(Gfap-luc)/FVB mice treated with IND24. Mice were inoculated with either RML[IND24] (green, n = 8; and light green, n = 5) or RML[IND24;IND24] (dotted green, n = 5). (B) Mean brain bioluminescence signal for mice in A, measured approximately every week; error bars represent SEM. Color scheme as in A. (C) PK-resistant PrPSc in CAD5 cells infected with RML[IND24;IND24] treated with 0–20 µM IND24, as indicated. PrPSc was assayed by immunoblot using HRP-conjugated HuM-D13 Fab. Images are representative of three replicates. Molecular weight markers of migrated protein standards represent 30 and 20 kDa. (D) Kaplan–Meier survival curves for passage of RML[IND24] or RML[IND24;V] into Tg(Gfap-luc)/FVB mice treated with vehicle. Mice were inoculated with either RML[IND24] (red, n = 8; and light red, n = 3) or RML[IND24;V] (dotted red, n = 7). (E) Mean brain bioluminescence signal for mice in D, measured approximately every week; error bars represent SEM. Color scheme as in D. (FK) PK-resistant PrPSc in CAD5 cells (F) and N2a cells (G) infected with RML[IND24;V] then treated with 0–4 µM IND24, as indicated. (HK) PK-resistant PrPSc in CAD5 cells infected with RML (H and J) and RML[IND24;IND24] (I and K) treated with 0–10 µM Compound B (H and I) or quinacrine (J and K). In FK, PrPSc was assayed by immunoblot using HRP-conjugated HuM-D13 Fab. Images are representative of three replicates. Molecular weight markers of migrated protein standards represent 30 and 20 kDa.
Fig. 5.
Fig. 5.
Effects of IND24 treatment on ME7 prion infection. (A) PK-resistant PrP from the brains of terminal animals infected with ME7 prions and treated with vehicle (red) or IND24 (green), as indicated. PrPSc was probed using HRP-conjugated HuM-P Fab. Molecular weight markers of migrated protein standards represent 30 and 20 kDa. This color scheme applies to B, C, and H. (B) Average ratio of monoglycosylated to diglycosylated PrPSc in ME7-infected Tg(Gfap-luc)/FVB mice treated with vehicle (n = 4) or IND24 (n = 5). Error bars represent SD. n.s. = not significant. (C) Conformational stability of ME7 prions from IND24-treated (n = 4) and vehicle-treated (n = 4) Tg(Gfap-luc)/FVB mice. Samples were exposed to increasing concentrations of GdnHCl (0–4 M, as indicated), then subjected to PK digestion. Points represent average percentage of PK-resistant PrP remaining at each concentration of GdnHCl, and error bars represent SD. (DG) PK-resistant PrP in CAD5 cells (D and E) and LD9 cells (F and G) infected with ME7 (D and F) or ME7[IND24] (E and G), and then treated with 0–20 µM of IND24, as indicated. PK-resistant PrP was assayed by immunoblot using HRP-conjugated HuM-D13 Fab. Images are representative of three replicates. Molecular weight markers of migrated protein standards represent 30 and 20 kDa. (H) Mean survival of mice infected with RML or ME7, then treated with vehicle and/or IND24, initiated at the timepoints indicated. Number of animals per experiment are shown. Error bars represent the SEM. ***P < 0.001, n.s. = not significant, Log-rank test.
Fig. 6.
Fig. 6.
Effects of IND24 treatment in Tg mouse models of prion disease. Kaplan–Meier survival curves of mice treated with vehicle (red) or IND24 (green) in (A) Tg4053 mice infected with RML (IND24: n = 9, vehicle: n = 8); (B) Tg1014 mice infected with RML (IND24: n = 9, vehicle: n = 4); (C) Tg1014 mice infected with sCJD(MM1) (IND24: n = 9, vehicle: n = 7); (D) Tg2669 mice infected with sCJD(MM1) (IND24: n = 7, vehicle: n = 9); (E and F) Tg1014 mice in E (IND24: n = 6, vehicle: n = 8) and Tg2669 mice in F (IND24: n = 8, vehicle: n = 9) infected with a 1:1,000 dilution of sCJD(MM1); (G) Tg152 mice infected with sCJD(VV2) (IND24: n = 9, vehicle: n = 9); and (H) Tg(ElkPrP)12584 mice infected with CWD(Elk) (IND24: n = 6, vehicle: n = 9).

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