FIGURE 4.
NMDA induces proteolytical processing of LRP1 in primary cultured cortical neurons. A (a), primary cultured cortical neurons were prepared from E15–E16 wild type mouse embryos. On DIV 5, neurons were pretreated with 1 μm DAPT for 2 h, and then 50 μm NMDA was added for the time indicated. DMSO was used as control (vehicle treatment). Whole cell lysates were prepared and analyzed by immunoblotting with antiserum directed against LRP1. Actin served as a loading control. b, primary cultured cortical neurons were prepared from E15–E16 wild type mouse embryos. On DIV 5, neurons were pretreated with 1 μm DAPT for 2 h, and then 50 μm NMDA, 50 μm AP5, 50 μm NMDA plus 50 μm AP5, or 50 μm N-methyl-l-aspartate was added for 12 h (UT, untreated control). Whole cell lysates were prepared and analyzed by immunoblotting with α-LRP1 antiserum. Actin served as a loading control. c, densitometric quantification of LRP1-CTF from b. Data are means ± S.E. (error bars) of five independent experiments. Statistical analysis was done by ANOVA (*, p < 0.05; **, p < 0.01; n.s., not significant). d, primary cultured cortical neurons were prepared from E15–E16 NesCreLRP1lox/lox (KO) and E15-E16 LRP1lox/lox (Control) mouse embryos. On DIV 5, control neurons were pretreated with 1 μm DAPT for 2 h, and then 50 μm NMDA was added for 12 h. KO neurons were pretreated with 1 μm DAPT for 2 h, and then 50 μm NMDA, 50 μm AP5, or 50 μm NMDA plus 50 μm AP5 was added for 12 h (UT, untreated control). Whole cell lysates were prepared and analyzed by immunoblotting with α-LRP1 antiserum. Actin served as a loading control. e, primary cultured cortical neurons were prepared from E15–E16 wild type mouse embryos. On DIV 5, neurons were pretreated with 1 μm DAPT for 2 h, and then 50 μm NMDA, 50 μm AP5, or 50 μm NMDA plus 50 μm AP5, 50 μm NMDA plus 1 μm ifenprodil, or 50 μm NMDA plus 10 μm ifenprodil was added for 12 h. DMSO was used as control (vehicle treatment). Whole cell lysates were prepared and analyzed by immunoblotting with α-LRP1 antiserum. Actin served as a loading control. B, primary cultured cortical neurons were prepared from E15 wild type mouse embryos. On DIV 5, neurons were pretreated with 10 μg/ml GST or 10 μg/ml GST-RAP for 30 min, and then 1 μm DAPT was added, followed by 50 μm NMDA for 12 h. Whole cell lysates were prepared and analyzed by immunoblotting with α-LRP1 antiserum. Actin served as a loading control. A representative blot (a) and quantitative analysis of four independent experiments (b) are shown. Error bars in b, S.E. Statistical analysis was done by ANOVA (*, p < 0.05). C, primary cultured cortical neurons were prepared from E15 wild type mouse embryos. On DIV 7, neurons were treated with 50 μm NMDA (N) for 4 h or left untreated (UT). After cross-linking with 1% paraformaldehyde, cell lysates were prepared, and DNA was sheared by sonification. Subsequently, chromatin immunoprecipitation was performed with an α-LRP1 antibody, rabbit IgG as negative control, or α-CREB as positive control. PCR amplification of c-fos promoter fragments was done with different primer sets to include transcription factor binding sites CRE −64, CRE −294, and CRE −342, and the serum-response element (SRE). The numbering refers to the fos gene in the Ensembl database (ENSMUSG00000021250).