Inhibition of Polo-like kinase 1 reduces beta-amyloid-induced neuronal cell death in Alzheimer's disease

Abstract

Alzheimer's disease (AD) is a progressive and fatal brain disease, but the pathogenesis of AD is still not understood. Aberrant cell-cycle re-entry of neuronal cells is emerging as a potential pathological mechanism in AD. Polo-like kinase 1 (Plk1) is an established regulator of many cell cycle-related events. Interestingly, Plk1 is present in susceptible hippocampal and cortical neurons of AD patients but not age-matched controls. However, whether Plk1 is involved in the pathogenesis of AD remains elusive. In this study, we showed that Plk1 activity is elevated in AD patient brain as indicated by the increased phosphorylation signal of p150Glued, a Plk1-specific substrate. Furthermore, we demonstrated that Plk1 is elevated during the cell-cycle re-entry of neuronal cells in an in vitro cell-culture model. Significantly, inhibition of Plk1 kinase activity or depletion of Plk1 by RNAi reduces β-amyloid (Aβ)-induced neuronal cell death. These results validate Plk1 as a possible target for AD therapy.

Figure 1. An increased phosphorylation level of p150^Glued in AD neurons

Hippocampal tissues of AD patients or age-matched controls were subjected to immunohistochemistry staining with phospho-specific antibodies against pS179-p150^Glued. Plk1 phosphorylates p150^Glued-S179 specifically [27].

Figure 2. Plk1 expression is elevated in Aβ-treated neuronal PC12 cells

(A) PC12 cells were differentiated by treatment with NGF for 3d, incubated with Aβ_25-35 (10 μM) for 24 h in the presence or absence of BI 2536 (10 nM), and harvested for Western blotting with antibodies against Plk1 and β-actin, a loading control. (B) Samples prepared in the same way as in (A) were subjected to anti-Plk1 IP/kinase assay using GST-Orc2 as a substrate [28], followed by autoradiography. IP: immunoprecipitation.

Figure 3. Plk1 is essential for neuronal cell death

(A) Inhibition of Plk1 reduces Aβ-induced neuronal cell death in PC12 cells. PC12 cells were treated with NGF for 3 d, followed by Aβ_25-35 or Aβ_25-35 + BI 2536 treatment for 24h. Cells were then incubated with 10 μg/ml propidium iodide (PI) for 10 min at 37–, washed with PBS, and harvested for immunofluorescence (IF). Cell death was assessed based on the principle that only the nuclei of cells with compromised plasma membranes will be stained with PI. (B) Inhibition of Plk1 reduces Aβ-induced DNA replication in PC12 cells. PC12 cells were treated as in (A), and subjected to BrdU incorporation assay to monitor DNA synthesis. * P<0.05.

Figure 4. Depletion of Plk1 prevents Aβ-induced cell death and DNA replication in neuronal PC12 cells

(A) Depletion of Plk1 in PC12 cells. One day after PC12 cells were differentiated with NGF, cells were infected with lentiviruses targeting nt1424 or 593 of Plk1, treated with Aβ_25-35 on day 4 of post-NGF treatment, and harvested for Western blotting. R1424 and R593 indicate two different targeting sequences on rat Plk1. (B) Cells described in (A) were subjected to cell death assay. (C) Cells described in (A) were subjected to BrdU labeling assay. *P<0.05.