Figure 1.
CentA1-Ras signaling mediates Aβ-induced loss of dendritic spines. A, Expression level of CentA1 in dissociated hippocampal neurons treated with Aβ(1–42). Dissociated hippocampal neurons from P0 rats were transfected with sh-SC or sh-CentA1 using electroporation, cultured for 3 d and treated with vehicle (Veh; final 0.01% NH4OH) or 1 μm Aβ for 48 h. The level of CentA1 was analyzed by Western blotting and β-actin was used as a loading control. Numbers under blots represent normalized CentA1 expression in four independent experiments. B, Immunoblot analysis of the expression level of CentA1 in organotypic hippocampal slices treated with Aβ(1–42). Slices were prepared from postnatal day 6 rats and cultured for 1–2 weeks, then treated with Veh or 1 μm Aβ for 48 h and 7 d, respectively. The level of CentA1 was analyzed by Western blotting and β-actin was used as a loading control. Numbers under blots represent normalized CentA1 expression in 15 (48 h) and 22 (7 d) independent experiments. C, Representative images of apical dendrites from CA1 pyramidal neurons in organotypic slices. Slices were prepared from postnatal day 6 rats and cultured for 1–2 weeks. Neurons were biolistically transfected with sh-SC or sh-CentA1 together with EGFP, treated with either vehicle or 1 μm Aβ (1–42) for 7 d, and imaged with two-photon laser scanning microscopy. Scale bar, 5 μm. D, Quantification of spine density in neurons treated as in C. Error bars indicate SEM. *p < 0.01, ANOVA followed by post hoc test using least significant difference. The number of neurons was 8, 11, 8, and 8, respectively. E, Spine density in apical dendrites of CA1 pyramidal neurons transfected with sh-SC or sh-CentA1 together with an empty vector (Vec) or human amyloid precursor protein (APP). The number of neurons was 5, 6, and 5, respectively. Error bars indicate SEM. F, Images of apical dendrites of CA1 pyramidal neurons expressing EGFP and Vec or WT-CentA1. Scale bar, 5 μm. G, Images of apical dendrites of CA1 pyramidal neurons expressing EGFP and Vec or dominant-negative Ras (HRasS17N, DN-Ras). Scale bar, 5 μm. H, Quantification of spine density in neurons expressing EGFP and Vec, WT-CentA1, or DN-Ras and treated with Veh or 1 μm Aβ for 1 week. *p < 0.01. The number of neurons was 18, 9, 8, 6, and 6, respectively. I, Spine density in apical dendrites of CA1 pyramidal neurons treated with Veh or 1 μm Aβ together with DMSO or 10 μm FTI-277 for 1 week. *p < 0.01. Number of neurons was 8, 5, 6, and 11, respectively. Error bars indicate SEM.