Fig. 2

Pharmacological inhibition of Pyk2 increases Tau phosphorylation independent of changes in GSK3β activity. A–D, Acute hippocampal slices (thickness, 400 μm) from 4.5–5.5-month-old PS19^0/+ animals were treated with 1 μM Pyk2 inhibitor (PF-719) for 2 h in oxygenated artificial CSF at room temperature and were homogenized in RIPA immediately following treatment. RIPA-soluble lysates were separated by SDS-PAGE and immunoblotted with the antibodies indicated. A, Representative immunoblot images of lysates from PF-719-treated acute hippocampal slices. Arrowhead indicates pGSK3β Y216. B–D, Quantification of A. PF-719 treatment successfully inhibited Pyk2 activity (pPyk2 Y402 normalized to total Pyk2). Pyk2 inhibition significantly increased the phosphorylation Tau at S202/T205 (AT8) normalized to total Tau (HT7) (D) while GSK3β activity (pGSK3β Y216 normalized to total GSK3β) remained unchanged (C). Data are graphed as mean ± SEM, unpaired two-tailed t-test, **p < 0.01, ****p < 0.0001, n = 8. E–I, iPSC-derived human cortical neurons (90–100 days post terminal differentiation) were treated with PF-719 at indicated concentrations for 2 h at 37 °C and, immediately following treatment, homogenized in RIPA containing 1% SDS. Lysates were separated by SDS-PAGE and immunoblotted with the antibodies listed. E, Representative immunoblot images of lysates from PF-719-treated iPSC-derived human cortical neurons. F–I, Quantification of E. PF-719 treatment significantly inhibited Pyk2 activity (pPyk2 Y402 normalized to total Pyk2) (F), while no changes in GSK3β activity (pGSK3β Y216 normalized to total GSK3β) were observed at any concentration of PF-719 (G). Pyk2 inhibition resulted in increased levels of Tau phosphorylation at S396/S404 (PHF-1) normalized to total Tau (HT7) (H) and S202/T205 (AT8) normalized to total Tau (HT7) (I) at every concentration of PF-719 administered. Data are graphed as mean ± SEM, one-way ANOVA with Dunnett’s multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 6