Research Paper Volume 12, Issue 24 pp 25581—25598

Figure 5. JMF3086 inhibited GSK3b-tau phosphorylation by activating Akt phosphorylation and decreasing neuronal apoptosis cascade in LRRK2-G2019S neurons. (A) Western blot of total and phosphorylated Akt, GSK3b, and tau after treatment with different concentrations of JMF3086. (B–D) Quantification of the effects of JMF3086 on the ratios of (B) p-Akt to total Akt, (C) p-GSK3α/β (Ser21/9) to total GSK3a/b, and (D) PHF-Tau to Tau1 in stably transfected LRRK2-G2019S SH-SY5Y cells. (B) The relative expression of p-Akt to total Akt in SH-SY5Y controls, LRRK2-WT, and LRRK2-G2019S after treatment with DMSO solvent, 0.05 μM, 0.1 μM, and 0.5 μM JMF3086 was 0.66±0.08, 0.63±0.07, 0.55±0.05, 0.56±0.06, 0.78±0.10, 0.89±0.09, and 0.91±0.10, respectively. P=0.04 for LRRK2-G2019S with DMSO solvent vs. LRRK2-G2019S with 0.05 μM JMF3086; P=0.03 for LRRK2-G2019S with DMSO solvent vs. LRRK2-G2019S with 0.1 μM JMF3086; P=0.03 for LRRK2-G2019S with DMSO solvent vs. LRRK2-G2019S with 0.5 μM JMF3086, all one-way ANOVA. (C) The relative expression of p-GSK3α/β (Ser21/9) to total GSK3a/b in SH-SY5Y controls, LRRK2-WT, and LRRK2-G2019S after treatment with DMSO solvent, 0.05 μM, 0.1 μM, and 0.5 μM JMF3086 was 0.32±0.07, 0.34±0.08, 0.20±0.08, 0.16±0.09, 0.55±0.10, 0.60±0.11, and 0.75±0.10, respectively. P=0.03 for LRRK2-G2019S with DMSO solvent vs. LRRK2-G2019S with 0.05 μM JMF3086; P=0.02 for LRRK2-G2019S with DMSO solvent vs. LRRK2-G2019S with 0.1 μM JMF3086; P=0.02 for LRRK2-G2019S with DMSO solvent vs. LRRK2-G2019S with 0.5 μM JMF3086, all one-way ANOVA. (D) The relative expression of PHF-Tau to Tau1 in SH-SY5Y controls, LRRK2-WT, and LRRK2-G2019S after treatment with DMSO solvent, 0.05 μM, 0.1 μM, and 0.5 μM of JMF3086 was 0.48±0.05, 0.79±0.10, 0.75±0.05, 0.65±0.08, 0.52±0.03, 0.50±0.06, and 0.49±0.07, respectively. P=0.02 for SH-SY5Y controls or LRRK2-WT vs. LRRK2-G2019S; P=0.04 for LRRK2-G2019S with DMSO solvent vs. LRRK2-G2019S with 0.05 μM JMF3086; P=0.03 for LRRK2-G2019S with DMSO solvent vs. LRRK2-G2019S with 0.1 μM JMF3086; P=0.03 for LRRK2-G2019S with DMSO solvent vs. LRRK2-G2019S with 0.5 μM JMF3086, all one-way ANOVA. All neurons were treated for 48 h and then lysed. Equal amounts of protein lysate were subjected to SDS-PAGE and the proteins e analyzed by Western blotting. Immunoblots were probed with the indicated antibodies. All experiments were repeated three times. Data represent mean ± SEM. *P<0.05, **P<0.01.