Hes1 overexpression blocked the effects of amyloid beta (Aβ) on the morphology, GABAergic connectivity and survival of cultured hippocampal neurons. (A-C) E17 hippocampal neurons were plated at a density of 40,000 cells/cm2 and cultured for 7 days in vitro (DIV). Neurons were treated with Aβ (5 μM), and/or they were co-transfected with enhanced green fluorescent protein (EGFP) and a myc-tagged Hes1 vector for 16 h. The cells were fixed and labeled with anti-EGFP, anti-myc and anti- vesicular inhibitory amino acid transporter (VIAAT) antibodies, and examined by immunofluorescence. (A) Representative micrographs of cultured neurons under different conditions. Nuclear labeling (in purple) corresponds to myc-tagged Hes1. Punctuated red dots correspond to VIAAT immunostaining. Lower panels show the boxed regions at higher magnification. (B) Morphometric analysis was performed as indicated in Materials and methods. Note that Aβ decreased the length of primary dendrites (left panel) and increased their number (right panel), whereas Hes1 induced opposite effects, counteracting those of Aβ. (C) Hes1 overexpression increased the number of GABAergic terminals in cultured neurons, overriding the decrease induced by Aβ administration. (D) 7 DIV neurons (30,000 cells/cm2) were treated with Aβ (5 μM). Two days later the neurons were co-transfected with EGFP and the myc-tagged Hes1 vector, and the cells were stained with anti-EGFP, anti-myc antibodies and 4',6-diamidino-2-phenylindole (DAPI) on the following day. EGFP and myc dual-labeled cells with intact nuclei were counted. Approximately 50% of transfected neurons survived Aβ treatment. *P < 0.05, **P < 0.01, and ***P < 0.001.
Chacón and Rodríguez-Tébar Alzheimer's Research & Therapy 2012 4:31 doi:10.1186/alzrt134