Figure 2. DB772 inhibits BVDV in primary sheep microglia (A) and Rov9 cells (B). Cells were inoculated with BVDV-containing PrPSc inoculum [55]. Following establishment of PrPSc accumulation, treatment groups were maintained in culture with 4 mM of DB772 for four passages. At the fourth passage (P-4, end of DB772 Tx), an aliquot of cells was lysed for BVDV antigen ELISA. All cultures were then continued for another four passages without DB772. Cells were collected for BVDV antigen ELISA at the end of those four clearance passages (P-8, end of clearance). A standard curve was used to transform the corrected optical densities into relative concentration of BVDV antigen. Data columns represent the means 6 one standard deviation. Results at each passage were statistically compared between DB772-treated and untreated groups. The y-axis reference line indicates the minimum detection limit of the ELISA. *, P,0.001. {, below assay detection limit. 1, one positive sample.
DB772 effect on PrPSc
To determine if DB772 inhibits PrP accumulation, microgliaSc/DB772, microgliaSc/UnTx, Rov9Sc/DB772, and Rov9Sc/UnTx cells were assayed for PrPSc levels. The same time points that were used for BVDV detection were used for PrPSc detection. Treatment with 4 mM DB772 reduced PrPSc levels in cell lysates below detectable limits (Fig. 3A). Based on the relative minimum detection limit of the PrPSc ELISA, as determined by the standard curves, this decrease in PrPSc at P-4 is at least 1.8 logs for microgliaSc/DB772 cells and 2.2 logs for Rov9Sc/DB772 cells. After four clearance passages without DB772 (P-8) one microgliaSc/ DB772 group and one Rov9Sc/DB772 group each contained minimal amounts of detectable PrPSc as determined by the standard curve (Fig. 3B). The ELISA used in this study has been commercially validated for regulatory use and it has also previously been shown in sheep microglial cells and Rov9 cells that positive and negative ELISA results correspond appropriately with positive and negative proteinase K-resistant immunoblotting results [49]. We reconfirmed this conclusion specifically in this set of experiments by immunoblotting after DB772 treatment. At passage five, DB772treated (4 mM) Rov9Sc/DB772 cell lysates lacked detectable proteinase K-resistant PrP, whereas the expected bands were detected in the Rov9Sc/UnTx cell lysate (Fig. 3C). As a control for the negative immunoblot signal, electrophoresed samples of PTAprecipitated lysates stained with SYPRO Ruby demonstrate the successful precipitation of proteins (Fig. 3D), confirming the specific loss of PrPSc in the DB772-treated samples.Scmulate detectable PrPSc (0/9 were PrPSc positive); whereas, Rov9 cells inoculated with the Rov9Sc/UnTx-derived lysate consistently accumulated PrPSc (9/9 were PrPSc positive); thus, importantly demonstrating the loss of prion infectivity. Results are the mean of three independent treatments, each conducted in triplicate.
DB772 effect on PRNP transcript and PrPC expression levels
Since PrPC expression is required for PrPSc accumulation, one potential mechanism of DB772-mediated inhibition of PrPSc accumulation could be inhibition of PrPC expression. PRNP transcript and total prion protein (PrP) levels were assayed in DB772-treated cells to determine if they were decreased as compared to untreated cells. Levels of PRNP transcript (Fig. 5A) and total PrP (Fig. 5B) were not decreased in microgliaSc/DB772 cells or in microgliaC/DB772 cells. In fact, at P-4 the PRNP transcript levels are significantly elevated in microgliaSc (P = 0.003) and microgliaC (P,0.012), although increased total PrP expression could only be verified in microgliaC (P,0.001). Similarly, no decrease in PRNP transcript levels was detected in Rov9 cells. The trend towards an increase in PRNP transcript was also evident in Rov9 cells; however, the magnitude of change was less as compared to microglial cells, and only one group (RovC/DB772) showed statistical significance (Fig. 5C). Total PrP levels similarly failed to show a decrease upon DB772 treatment (Fig. 5D). In summary, expression of PrPC was not inhibited in DB772-treated microglial cells or Rov9 cells.
Concentration response of DB772 on pestivirus and PrPSc DB772 effect on prion infectivity
A prion is ultimately defined by its infectious capability; thus, to confirm that the reduced PrPSc levels correlated with reduced prion infectivity, we compared prion infectivity between Rov9Sc/ DB772 cultures and Rov9Sc/UnTx cultures (Fig. 4). To confirm the concentration dependence of the anti-PrPSc and anti-pestiviral effects, and to compare these concentration-dependent effects, microgliaSc and Rov9Sc cells were exposed to a range of DB772 concentrations, and the relative levels of PrPSc and pestivirus were determined. The anti-PrPSc and anti-pestiviral effects were concentration dependent.Figure 3. DB772 inhibits PrPSc accumulation in primary sheep microglial cells and Rov9 cells. Determination of PrPSc levels in sheep microglial cells (A) and Rov9 cells (B). Cells were inoculated with PrPSc, and following establishment of PrPSc accumulation, treatment groups were maintained in culture with 4 mM of DB772 for four passages. At the fourth passage (P-4, end of DB772 Tx), an aliquot of cells was lysed for PrPSc ELISA. All cultures were then continued for another four passages without DB772. Cells were collected for PrPSc ELISA at the end of those four passages (P-8, end of clearance). A standard curve was used to transform the corrected optical densities into relative concentration of PrPSc. Data columns represent the means 6 one standard deviation. Results at each passage were statistically compared between DB772-treated and untreated groups. The y-axis reference line indicates the minimum detection limit of the ELISA. *, P,0.05. Positive and negative ELISA results were compared to proteinase K digestion-based immunoblotting (C). Passage 5 Rov9Sc/UnTx and Rov9Sc/DB772 samples were lysed, treated with 50 mg/ml of proteinase K, precipitated with phosphotungstic acid, and immunoblotted using the monoclonal anti-PrP antibody F99/97.6.1. Proteinase K-resistant prion protein bands were detected in P-5 Rov9Sc/UnTx samples, whereas bands were not detected in the P-5 Rov9Sc/DB772 lysates, consistent with the ELISA results. Successful precipitation of protein via the phosphotungstic acid method was confirmed in Rov9Sc/DB772 and Rov9Sc/UnTx samples, with most protein being proteinase K susceptible (D). The positions of molecular mass standards (in kilodaltons) are indicated to the left of the immunoblot (C) and gel (D).(Table 2) the dynamic range for anti-pestiviral activity was determined to be 0.004?.04 mM in microgliaSc and 0.0004?0.004 mM in RovSc, whereas the dynamic range for PrPSc inhibition in microgliaSc and in RovSc was 0.4?.0 mM (Table 2). Since the goal of this study was not to re-describe the antipestiviral effects of this compound [56], subsequent independent experiments focused solely on the anti-PrPSc concentration range. The final anti-PrPSc 50% tissue culture effective concentrations (TCEC50) for microgliaSc and for Rov9Sc cells were 2.460.2 mM and 1.960.4 mM, respectively (Table 2). This would represent a greater than two-log difference between the anti-PrPSc and antipestiviral TCEC50 values. In further independent experiments at higher concentrations, to describe the anti-PrPSc concentration dependence, complete anti-pestivirus activity was consistently measured at 0.4 mM (the lowest concentration used); thus, the anti-pestivirus TCEC50 must be lower than 0.4 mM and is
different from the anti-PrPSc TCEC50 of 2.4 mM and 1.9 mM (microglialSc and Rov9Sc, respectively) (Table 2).
