Low density lipoprotein receptor-related protein 1 promotes anti-apoptotic signaling in neurons by activating Akt survival pathway - PubMed (original) (raw)

Rodrigo A Fuentealba et al. J Biol Chem. 2009.

Abstract

The low density lipoprotein receptor-related protein 1 (LRP1) is a multi-ligand receptor abundantly expressed in neurons. Previous work has shown that brain LRP1 levels are decreased during aging and in Alzheimer disease. Although mounting evidence has demonstrated a role for LRP1 in the metabolism of apolipoprotein E/lipoprotein and amyloid-beta peptide, whether LRP1 also plays a direct role in neuronal survival is not clear. Here, we show that LRP1 expression is critical for the survival of primary neurons under stress conditions including trophic withdrawal, the presence of apoptosis inducers, or amyloid-beta-induced neurotoxicity. Using lentiviral short hairpin RNA to knock down endogenous LRP1 expression, we showed that a depletion of LRP1 leads to an activation of caspase-3 and increased neuronal apoptosis, an effect that was rescued by a caspase-3 inhibitor. A correlation between decreased Akt phosphorylation and the activation of caspase-3 was demonstrated in LRP1 knocked down neurons. Notably, LRP1 knockdown decreased insulin receptor levels in primary neurons, suggesting that decreased neuronal survival might be a consequence of an impaired insulin receptor signaling pathway. Correspondingly, both insulin receptor and phospho-Akt levels were decreased in LRP1 forebrain knock-out mice. These results demonstrate that LRP1 mediates anti-apoptotic function in neurons by regulating insulin receptor and the Akt survival pathway and suggest that restoring LRP1 expression in Alzheimer disease brain might be beneficial to inhibiting neurodegeneration.

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Figures

FIGURE 1.

FIGURE 1.

Knockdown of LRP1 in primary neurons. A, titration of LRP1 shRNA lentiviruses targeting two different regions of LRP1. The neurons were infected with 1 × 105, 2.5 × 105, or 1 × 106 transforming units of the indicated lentiviruses, and LRP1 and β-actin levels were analyzed by Western blot. A lentivirus prepared from pLKO.1 empty vector was used as a control. Both LRP1 shRNAs efficiently decreased LRP1 levels. LRP1 shRNA 2 was selected for the remainder of this study. B, triple staining for LRP1 (red), MAP2 (green), and DAPI (blue) in control and LRP1 shRNA-infected neurons. A robust LRP1 staining was detected in the soma and a spotted pattern in MAP2-positive processes and were significantly decreased upon infection with LRP1 shRNA. Scale bar, 50 μm.

FIGURE 2.

FIGURE 2.

LRP1 knockdown decreases cell viability in primary neurons upon trophic withdrawal and Aβ42-induced toxicity. A, effect of control or LRP1 shRNA on neuronal viability analyzed by the MTT reduction assay in the presence or absence of B27 supplement. Increased neuronal cell death was detected in LRP1 shRNA-infected neurons upon trophic support withdrawal. The mean differences were compared by ANOVA and Dunnett's test using control infection + B27 supplement cells as the reference group (**, p < 0.01) or by ANOVA and Bonferroni's test for selected groups (##, p < 0.01). B, LRP1 knockdown renders neurons susceptible to Aβ-induced toxicity. Primary neurons were infected with control or LRP1 shRNA and incubated with 0, 0.1, 1, and 10 μ

m

Aβ42 for 18 h, and neuronal viability was assessed by reduction of the MTS redox dye. The mean differences were compared by unpaired Student's t test. *, p < 0.05; **, p < 0.01.

FIGURE 3.

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.

FIGURE 4.

FIGURE 4.

LRP1 regulates Akt phosphorylation in vitro and in vivo. A, decreased phospho-Akt and phospho-GSK-3β levels in LRP1 knocked down neurons upon trophic withdrawal. Primary neurons were infected with control or LRP1 shRNA, and the levels of LRP1, phospho-Akt (Ser473), total Akt, and phospho-GSK-3β were analyzed by Western blot under both control conditions and upon 18 h of treatment with neurobasal-only media. The β-actin levels were determined as a loading control. The levels of phospho-Akt and phospho-GSK-3β were additionally decreased in LRP1 knocked down neurons upon trophic withdrawal. B, protein levels from experiments in A were determined by densitometry, and the corresponding phospho-Akt to total Akt and phospho-GSK-3β to β-actin ratios were calculated and plotted relative to control neurons. The mean differences were compared by ANOVA and Dunnett's test using control infection + B27 supplement treated cells as the reference group (**, p < 0.01; *, p < 0.05) or by ANOVA and Bonferroni's test for selected groups (#, p < 0.05). C, decreased phospho-Akt in LRP1 forebrain knock-out mice. Equal amounts of total brain homogenates (40 μg) from LRP1 forebrain knock-out mice and littermate controls were subjected to Western blot analysis, and both phospho-Akt (Ser473) and total Akt levels were determined (n = 4). Lower panel, densitometric analysis of phospho-Akt and total Akt levels determined from experiments as in A. The mean differences were compared by unpaired Student's t test, * p < 0.05. WT, wild type.

FIGURE 5.

FIGURE 5.

Decreased insulin receptor levels in LRP1 knocked down neurons and in LRP1 forebrain knock-out mice. Primary neurons were infected with control or LRP1 shRNA, and insulin receptor (IR) and PI3K-p85 levels were determined by Western blot under both control conditions (A) or upon 18 h of treatment with neurobasal-only media (B). Left panels, protein levels of the insulin receptor-β subunit from experiments in A and B were determined by densitometry and plotted relative to control neurons. The levels of the insulin receptor, but not of PI3K-p85, were decreased in LRP1 knocked down neurons under both basal and trophic withdrawal conditions. The mean differences were compared by unpaired Student's t test. *, p < 0.05; **, p < 0.01. C, decreased insulin receptor in LRP1 forebrain knock-out mice. Equal amounts of total brain homogenates (40 μg) from LRP1 forebrain knock-out mice and littermate controls were subjected to Western blot analysis, and the levels of both insulin receptor and β-actin were determined. Representative results from n = 4 are shown. Left panel, densitometric analysis showing the insulin receptor-to-β-actin ratio determined from experiments in C. The mean differences were compared by unpaired Student's t test. *, p < 0.05. WT, wild type.

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