Tetracyclines affect prion infectivity

Abstract

Prion diseases are transmissible neurodegenerative disorders of humans and animals for which no effective treatment is available. Conformationally altered, protease-resistant forms of the prion protein (PrP) termed PrP(Sc) are critical for disease transmissibility and pathogenesis, thus representing a primary target for therapeutic strategies. Based on previous findings that tetracyclines revert abnormal physicochemical properties and abolish neurotoxicity of PrP peptides in vitro, we tested the ability of these compounds to interact with PrP(Sc) from patients with the new variant of Creutzfeldt-Jakob disease (vCJD) and cattle with bovine spongiform encephalopathy (BSE). The incubation with tetracycline hydrochloride or doxycycline hyclate at concentrations ranging from 10 microM to 1 mM resulted in a dose-dependent decrease in protease resistance of PrP(Sc). This finding prompted us to investigate whether tetracyclines affect prion infectivity by using an animal model of disease. Syrian hamsters were injected intracerebrally with 263K scrapie-infected brain homogenate that was coincubated with 1 mM tetracycline hydrochloride, 1 mM doxycycline hyclate, or vehicle solution before inoculation. Hamsters injected with tetracycline-treated inoculum showed a significant delay in the onset of clinical signs of disease and prolonged survival time. These effects were paralleled by a delay in the appearance of magnetic-resonance abnormalities in the thalamus, neuropathological changes, and PrP(Sc) accumulation. When tetracycline was preincubated with highly diluted scrapie-infected inoculum, one third of hamsters did not develop disease. Our data suggest that these well characterized antibiotics reduce prion infectivity through a direct interaction with PrP(Sc) and are potentially useful for inactivation of BSE- or vCJD-contaminated products and prevention strategies.

Fig 1.

Tetracyclines revert the protease resistance of PrP^Sc from vCJD brains. Immunoblot analysis of partially purified PrP^Sc from vCJD after incubation in the absence (Ctr) or presence of tetracycline (A) and doxycycline (B), followed by proteinase K digestion. The blots were probed with the antibody 3F4 (1:50,000). Molecular mass markers are indicated to the left. (C) Quantification of the protease-resistant core of PrP^Sc in tetracycline- (Tetra) and doxycycline-treated (Doxy) samples. The values were obtained by densitometric analysis of immunoblots and are expressed as percentage of signal intensity of samples nontreated with tetracyclines. The data are the mean ± SEM of six experiments from three different patients. °, P < 0.05, and *, P < 0.01 versus the relevant control group (Dunnett's test).

Fig 2.

Effects of tetracyclines on survival of scrapie-infected Syrian hamsters. (A) Survival time of hamsters injected intracerebrally with a 10⁻⁵ dilution of 263K scrapie-infected brain homogenate (shaded area) or the same inoculum after incubation with doxycycline (white area). (B) Survival time of hamsters injected intracerebrally with a 10⁻⁶ dilution of 263K scrapie-infected brain homogenate (shaded area) or with the same inoculum after incubation with tetracycline (white area).

Fig 3.

Effects of tetracycline on cerebral MRI changes in scrapie-infected hamsters. T2-weighted images of hamsters injected intracerebrally with a 10⁻⁶ dilution of normal brain homogenate (A), 263K scrapie-infected brain homogenate (B), or 263K scrapie-infected brain homogenate after incubation with tetracycline (C) are shown. The MRI was carried out 90 days after inoculation. Note the striking difference in thalamic hyperintensity between tetracycline-untreated (B) and tetracycline-treated (C) hamsters.

Fig 4.

Effects of tetracycline on accumulation of PrP^Sc in the brain of scrapie-infected hamsters. Shown are PrP immunoblot analysis and immunohistochemistry of brain tissue of three hamsters (1–3) from the tetracycline-untreated (Upper) and tetracycline-treated (Lower) group with the antibody 3F4. The animals, randomly selected before infection, were injected intracerebrally with a 10⁻⁶ dilution of 263K scrapie-infected brain homogenate (Upper) or 263K scrapie-infected brain homogenate after incubation with tetracycline (Lower) and killed 90 days after inoculation (experiment 3).