Fig 2: AZ59 reduces PrPC level and reverses aberrant eEF2 and JNK signaling in APP/PS1 mice. (A) Representative immunoblots of synaptosomal PrPC, PSD-95 and ß-Actin from brains of 14-16-month APP/PS1 mice after 1 week of antibody treatment at 100 mg/kg. (B) Quantification of (A). One week (two doses) of treatment at 100 mg/kg significantly reduced levels of synaptic PrP relative to PSD-95 (*,P < 0.05). Analysis by two-tailed t-test. n = 13 mice per group, data are mean ± SEM. (C) Representative western blots of TBS insoluble, Triton X-100 soluble total, and phospho-eEF2 (T56) from hippocampi of 14-month wild type and APP/PS1 mice after 5 weeks of treatment. (D) Quantification of C. There was a significant increase in the level of Triton X-100 soluble phospho-eEF2 (T56) normalized to total eEF2 in hippocampi of 14-month APP/PS1 mice treated with IgG control compared to wild type controls (*,P < 0.05). There was no significant difference in phospho-eEF2 (T56) between 14-month wild type controls and AZ59 treated APP/PS1 mice. Analysis by one-way ANOVA with Dunnett's multiple comparisons test. n = 7–24 mice per group, data are mean ± SEM. (E) Representative western blots of TBS insoluble, Triton X-100 soluble total, and phospho-JNK (T183/Y185) from hippocampi of 14-month wild type and APP/PS1 mice after 5 weeks of antibody treatment. (F) Quantification of (E). There was a significant increase in the level of Triton X-100 soluble phospho-JNK (T183/Y185) normalized to total JNK of the larger JNK isoform (upper band) in hippocampi of 14-month APP/PS1 mice treated with IgG control compared to wild type controls (**,P < 0.01). There was no significant difference in phospho-JNK (T183/Y185) of this isoform between wild type controls and APP/PS1 mice treated at 20 mg/kg. There was a significant increase in the level of phospho-JNK (T183/Y185) normalized to total JNK of the smaller JNK isoform (lower band) in aged APP/PS1 mice treated with IgG control compared to wild type controls (***,P < 0.001). There was no significant difference in phospho-JNK (T183/Y185) of this isoform between wild type controls and APP/PS1 animals treated with AZ59 at 20 mg/kg. Analysis by one-way ANOVA with Dunnett's multiple comparisons test. n = 7–24 mice per group, data are mean ± SEM.

Fig 3: AZ59 does not alter glial activation or Aß load. (A) Representative images of immunofluorescent staining for Iba1 (red) and CD68 (green) from the hippocampus of 14-month-old WT and APP/PS1 after 5 weeks of treatment. Scale bar = 60 µm. (B–C) Quantification of Iba1 (B) and CD68 (C) immunoreacted area. (*,P < 0.05; **, P < 0.01) n = 12–13 mice per group, data are mean ± SEM, one-way ANOVA with Dunnett's comparison to APP/PS1 control IgG. (D) Representative images of anti-GFAP immunostaining taken in the hippocampus of 14-month-old WT and APP/PS1 after 5 weeks of treatment. Scale bar = 60 µm. (E) Quantification of GFAP immunoreacted area. (**, P < 0.01) n = 12–13 mice per group, data are mean ± SEM, one-way ANOVA with Dunnett's comparison to APP/PS1 control IgG. (F) Representative images of ThioS staining in the hippocampus of 14-month-old WT and APP/PS1 after 5 weeks of treatment. Scale bar = 200 µm. (G) Quantification of ThioS plaque area. No significance between the groups (P > 0.05) n = 12–13 mice per group, data are mean ± SEM, one-way ANOVA with Dunnett's comparison to APP/PS1 control IgG. (H) Aßo from TBS homogenate of APP/PS1 mouse cortex captured by PrPC(23-230) on a 384-well plate. No significance between the groups (P > 0.05) n = 6 mice per group, data are mean ± SEM, one-way ANOVA with Tukey's multiple comparison's test.

Fig 4: AZ59 disrupts the Aßo-induced interaction of PrPC and mGluR5. (A) The binding of Myc-tagged fly-trap domain of mGluR5 to a 384-well plate coated with PrPC(23-230). Data are mean ± SEM, n = 3 replicates per sample. (B) Co-immunoprecipitation of HEK cell lysates expressing PrPC and Myc-mGluR5 pretreated with either vehicle, 6D11, AZ59, or control IgG before Aßo treatment. Membrane proteins were extracted with RIPA buffer and immunoprecipated with either a-PrPC or a-Myc. (C) Quantification of PrPC signal in (B) of a-Myc immunoprecipitate. Data are mean ± SEM, n = 3 replicates per sample. (D) Quantification of Myc signal in (B) of a-PrPC immunoprecipitate. Data are mean ± SEM, n = 3 replicates per sample.

Fig 5: AZ59 penetrates the blood-brain barrier at nanomolar concentrations. (A) Standard curves used to determine concentration of samples. 384-well plates were coated with PrPC(23-230) to capture AZ59 recovered from TBS and RIPA lysates from the cortex of treated mice. (B) AZ59 was added to total brain homogenate at a final concentration of 20 nmol/L and incubated overnight at 4C. TBS and RIPA fractions were collected and compared to a standard curve to determine the final concentration in each fraction. (C–D) AZ59 recovered from TBS (C) and RIPA (D) lysate from cortex of mice treated with the indicated mg/kg doses of AZ59 for either 5 or 6 weeks, and sacrificed 7 days after the last dose. Data are mean ± SEM, n = 5–6 per group, all samples were run in triplicate. (E) AZ59 recovered from serum of treated mice. Data are mean ± SEM, n = 7–10 per group, all samples were run in triplicate. (F) Table summarizing results in (C–E). Total brain concentration is the sum of TBS and RIPA fraction concentrations. Data are mean ± standard deviation. N/A, not assayed.