FIGURE 3.

LRP1 regulates apoptosis in primary neurons. A, neurons were infected with control or LRP1 shRNA, and apoptosis was analyzed by TUNEL assay in neurons treated for 18 h with neurobasal-only medium. Left panel, representative images showing nuclear staining of DNA (DAPI) or single and double strand DNA breaks (TUNEL). Positive controls from an untreated culture incubated with DNase-I are shown. LRP1 knockdown increases apoptosis in primary neurons. Right panel, quantification of TUNEL staining. TUNEL-positive neurons were counted blindly from at least 500 neurons from two fields of three independent experiments. B, primary neurons were infected with control or LRP1 shRNA and cleaved caspase-3, full-length caspase-3, LRP1, and β-actin levels were analyzed by Western blot upon 18 h of treatment in neurobasal-only media. Active caspase-3 is increased in LRP1 knocked down neurons. Each lane represents the result obtained from an independent infection. C, primary neurons were infected with control or LRP1 shRNA and were subjected to trophic withdrawal in the presence of vehicle control (dimethyl sulfoxide, DMSO) or the caspase-3 inhibitor z-DEVD-fmk for 18 h, and cell viability was analyzed by MTT assay. Caspase-3 inhibition decreases cell death in LRP1 knocked down neurons. The mean differences were compared by ANOVA and Dunnett's test using control infection + Me₂SO-treated cells as the reference group (*, p < 0.05) or by ANOVA and Bonferroni's test for selected groups (#, p < 0.05). The effects of LRP1 knockdown on neuronal cell death were rescued in the presence of the caspase-3 inhibitor.