Figure 5.

Tranforming growth factor β1 (TGFβ1) activates nuclear factor kappa-B (NF-κB) and induces Hes1 expression, controlling dendrite morphology and GABAergic connectivity, and conferring cultured neurons with resistance to amyloid beta (Aβ) neurotoxicity. (A) Dual-luciferase NF-κB reporter gene assay. After transfecting NF-κB-luc and RL-TK-luc, 7 days in vitro (DIV) hippocampal neurons were incubated for 16 h and the neurons were then exposed to Aβ (5 μM) with TGFβ1 (10 ng/ml) added to the medium 1 h later. The neurons were analyzed after a further 4 h incubation and the data represent the means of six experiments. (B) Cultured neurons (7 DIV, 300,000 cells) were treated with Aβ (5 μM) and then TGFβ1 (10 ng/ml) 1 h later, and the cells incubated for a further 16 h. After lysing the cells, Hes1 expression was quantified by real time PCR (the means of 10 determinations are shown). (C-E) Hippocampal neurons (7 DIV, 40,000 cells/cm²) were transfected with enhanced green fluorescent protein (EGFP) and treated with Aβ (5 μM) and/or TGFβ1 (10 ng/ml). After 16 h, immunostaining for EGFP and vesicular inhibitory amino acid transporter (VIAAT) was performed as described in Materials and methods. (C) Representative micrographs of cultured neurons under different conditions (the lower panels show the boxed regions at higher magnification). (D) Morphometric analysis of Aβ- and TGFβ1-treated neurons. TGFβ1 increased the length (left panel) and decreased the number (right panel) of primary dendrites, thereby counteracting the effects of Aβ. (E) Quantification of GABAergic clusters. TGFβ1 increased the number of GABAergic terminals in cultured neurons and overrode the decrease in GABAergic terminals induced by Aβ. (F) Hippocampal neurons (7 DIV, 30,000 cells/cm²) were treated with Aβ (5 μM) and/or TGFβ1 (10 ng/ml) for 90 h and the number of intact nuclei were counted after 4',6-diamidino-2-phenylindole (DAPI) staining. TGFβ1 prevented Aβ-induced death of a portion of cultured hippocampal neurons. *P < 0.05, **P < 0.01, and ***P < 0.001.