ROS scavengers and calpain inhibitors are beneficial to prion-infected COCS and mice.

<p>(<b>A</b>) Lower graph: COCS were inoculated with RML or NBH, cultured for 21 dpi, and then treated with various compounds for 22 days. Upper graph: COCS were cultured for 14 days, incubated with POM1 and treated with the same compounds for 10–14 days. Viability (NeuN) was normalized to COCS exposed to IgG and to non-infectious brain homogenate (upper and lower gray dots, respectively). Calpain inhibitors (Calpeptin), antioxidants (Ascorbate, Isoascorbate, NaC), and superoxide dismutase mimetics (MnTBAP) were neuroprotective in both paradigms (red dots), whereas inhibition of AMPA, kainate receptors (CNQX), NMDA receptors (MK801), a mitochondrial membrane permeability transition pore (methazolamide), caspases (ZVAD), and of ER calcium stores (Dantrolene), (black dots) was ineffective; <i>n</i> = 9 biological replicates. The effects of compounds labeled with “†” on POM1-exposed COCS and zVAD labeled with “§” on RML-infected COCS were reported previously [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004662#ppat.1004662.ref015" target="_blank">15</a>], [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004662#ppat.1004662.ref013" target="_blank">13</a>] and are reproduced here for convenience. (<b>B</b>) COCS were exposed to NBH (-) or to RML (+) as in (A), and harvested at 39 dpi (<i>n</i> = 3). Prion titers were determined by SCEPA. No reduction in prion infectivity was observed in COCS treated with ascorbate (Asc) or NaC (<i>n</i> = 3 biological replicates). (<b>C</b>) Prion-infected COCS were treated with Asc. Protection was discernible for ≥53 dpi (red bars). Data were analyzed using a two-tailed t-test; <i>n</i> = 9 biological replicates. (<b>D</b>) Survival curves of prion-infected <i>tg</i>a<i>20</i> mice treated with AcHyT (median 95dpi) or vehicle (drinking water; median 88.5dpi). AcHyT treatment increased the incubation time of prion-infected mice (p = 0.0287). Results were analyzed using a Mantel-Cox log-rank test; <i>n</i> = 6 untreated mice and <i>n</i> = 7 AcHyT-treated mice. (<b>E</b>) COCS were treated with antioxidants from 21 dpi onwards (<i>n</i> = 3) and harvested at 40 dpi. Cleaved α-fodrin bands (145 and 150 kDa) were quantified and normalized to GAPDH (right). None of the antioxidants suppressed the prion-induced enhancement of α-fodrin cleavage; <i>n</i> = 3 biological replicates. (<b>F</b>) COCS were cultured for 14 days, treated with scFvPOM1 (400nM) or scFvPOM1 preincubated with recombinant PrP (1200nM) in presence of ascorbate, and harvested at 3 dpe. Cleaved α-fodrin bands (145 and 150 kDa) were quantified and normalized to calnexin (right). Ascorbate did not suppress the GDL induced enhancement of α-fodrin cleavage; <i>n</i> = 3 biological replicates.