FIGURE 5.

Expression of Lphn1 in neurons results in increased apoptosis. (A) Western blot of lysate from HEK293 cells that were contransfected with HA-Lphn1-GFP and one of four different Lphn1 shRNA plasmids or scrambled control plasmid (n = 1). The samples in these western blots that were immunoblotted with anti-HA, anti-Lphn1-p120, and anti-GFP antibodies were quantified and normalized against actin and the scrambled control plasmid, supporting strongest Lphn1 knockdown with the Lphn1 shRNA1, -3 and -4 plasmids. Molecular weights are as follows: HA-Lphn1-GFP = 125 kDa; βActin = 42kDa. (B) Representative photographs of caspase-3 (red) positive cells in wild-type and Cntn6^-/- neuronal cultures transfected with an EGFP expression vector (green) and either a Lphn1 shRNA3 or -4 plasmid or a scrambled control plasmid. DAPI staining is in blue. Arrowheads indicate caspase-3 expression in the soma of neurons. The scale bar indicates 100 μm. (C) Cotransfection of Lphn1 shRNA3 and -4 plasmids with an EGFP expression vector in wild-type primary cultures did not result in apoptosis difference compared to the scrambled control plasmid. However, apoptosis in Cntn6^-/- primary cultures was significantly reduced after treatment with Lphn1 shRNA3 and -4 compared to the scrambled control plasmid. About 60 transfected neurons were analyzed for caspase-3 immunoreactivity per condition of each independent experiment (n = 5 for Cntn6^+/+ cultures and n = 4 for Cntn6^-/- cultures). (D,E) In vivo analysis of caspase-3 expression (green) in the visual cortex of P14 wild-type and Cntn6^-/- animals revealed a significant increase of apoptosis in the absence of Cntn6. DAPI staining is in blue. Scale bar represents 150 μm. Analysis was performed on at least three sections per brain of wild-type and Cntn6^-/- mice (n = 5 per genotype). Statistical analyses were performed using unpaired Student’s t. The graph bars are presented as mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01.