Prion strain- and species-dependent effects of antiprion molecules in primary neuronal cultures

Abstract

Transmissible spongiform encephalopathies (TSE) arise as a consequence of infection of the central nervous system by prions and are incurable. To date, most antiprion compounds identified by in vitro screening failed to exhibit therapeutic activity in animals, thus calling for new assays that could more accurately predict their in vivo potency. Primary nerve cell cultures are routinely used to assess neurotoxicity of chemical compounds. Here, we report that prion strains from different species can propagate in primary neuronal cultures derived from transgenic mouse lines overexpressing ovine, murine, hamster, or human prion protein. Using this newly developed cell system, the activity of three generic compounds known to cure prion-infected cell lines was evaluated. We show that the antiprion activity observed in neuronal cultures is species or strain dependent and recapitulates to some extent the activity reported in vivo in rodent models. Therefore, infected primary neuronal cultures may be a relevant system in which to investigate the efficacy and mode of action of antiprion drugs, including toward human transmissible spongiform encephalopathy agents.

FIG. 1.

High sensitivity of postmitotic neurons to prion infection. (A) Accumulation of PrP^res in CGN^Ov cultures following infection with serial dilutions of 127S brain homogenate (final concentrations as indicated). De novo PrP^res was quantified at 28 days postexposure by densitometric image analysis of immunoblots in two independent experiments (mean ± standard deviation) and is expressed as a percentage of total PrP^res detected by immunoblotting of 1 mg of mouse brain from terminally ill 127S-infected tg338 mice. (B) CGN^Ov cultures were exposed to infectious prions either 2 or 8 days after plating, i.e., before or after their full differentiation. PrP^res accumulation was assessed at 30 days postplating by immunoblotting of PK-treated lysate using biotinylated monoclonal antibody ICSM18 (see methods).

FIG. 2.

Accumulation of PrP^res in CGN cultures upon exposure to rodent and human prions. CGN cultures were established from transgenic mice expressing mouse (A), hamster (B) or human (C) PrP and were exposed to brain homogenates (A and B) or purified PrP^Sc (C) from terminally ill mice infected with prions. CGN cultures established from PrP^0/0 transgenic mice (CGN^0/0) were also exposed to infectious prions in parallel. (A) CGN^Mo cultures exposed to mouse strain 139A, Fukuoka-1 (FK), 22L, or ME7 at a final concentration of 0.1% (wt/vol). (B) CGN^Ha cultures exposed to hamster strain Sc237 at a final concentration of 0.002%. (C) CGN^Hu cultures exposed to type 1 CJD (T1 CJD) at a final concentration equivalent to 0.1% of brain homogenate. The data shown in panels B and C correspond to duplicate culture wells within a representative experiment. In all PrP-expressing cultures, PrP^res accumulation increased from 14 to 28 days postexposure (d.p.e.) and was weak or absent in nonpermissive CGN^0/0 cultures. Cell lysates were PK treated, and PrP^res was detected by immunoblotting using biotinylated monoclonal antibody ICSM18 (A and C) or Sha31 (B).

FIG. 3.

Effect of chlorpromazine, Congo red, and MS-8209 on sheep prion propagation in CGN^Ov cultures at an early or advanced stage of infection. (A) CGN^Ov cultures were infected on day 2, two treatments (chlorpromazine, 5 μM; Congo red, 7 μM; MS-8209, 7.5 μM; or solvent, 0.1%) were performed on days 5 and 8, and cells were lysed on day 12. Control wells of CGN^Ov and nonpermissive CGN^0/0 cultures were infected similarly and left untreated (Control). (B and C) CGN^Ov cultures were infected on day 3, drugs (B, Congo red at 1.5 μM and 7 μM; C, 7.5 μM MS-8209 or 0.1% solvent) were added 1 day or 11 days after prion exposure and then twice a week; cells were lysed on day 21 (respectively, after 5 or 2 treatments). PrP^res was revealed by immunoblotting of PK-treated lysates using biotinylated monoclonal antibody ICSM18. d.p.e., days postexposure.

FIG. 4.

Inhibition of PrP^res accumulation by chlorpromazine, Congo red, and MS-8209 in CGN cultures infected by prions from different species. CGN cultures expressing mouse (A), hamster (B), or human (C) PrP^C were infected by brain homogenates on day 2 with prions from the corresponding species: 139A (0.01% final concentration), Sc237 (0.01%), and type 1 CJD (T1 CJD; 0.05%). (C) Nonpermissive CGN^0/0 cultures were also exposed to T1 CJD prions. Three treatments were performed on days 5, 8, and 11 with chlorpromazine at a concentration of 1 μM (B) or 5 μM (A and C), Congo red (7 μM), or MS-8209 at a concentration of 3.5 μM (B) or 7.5 μM (A and C), and cells were lysed on day 15. PrP^res was revealed by immunoblotting of PK-treated lysates using biotinylated monoclonal antibody Sha31 (A and B) or ICSM18 (C).

FIG. 5.

Comparison of antiprion activity of polyene antibiotic MS-8209 according to hamster prion strains. CGN cultures expressing hamster PrP were infected with hamster prion strains Sc237 (A; 0.01% final concentration) or 139H (B; 0.002%) on day 2. Three treatments with MS-8209 (3.5 μM) were performed, starting at the early stage of infection, and cells were lysed shortly after the last treatment. PrP^res was revealed by immunoblotting of PK-treated lysates using biotinylated monoclonal antibody Sha31. (C) PrP^res inhibition was quantified by densitometric image analysis of immunoblots (n = 3 independent experiments) and is expressed as a percentage of controls (mean ± standard error of the mean). MS-8209 treatment was significantly more effective toward Sc237 hamster prions than the 139H strain. *, P< 0.05 (Student's t test).