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Comparative Study
. 2006 Oct 25;26(43):10949-57.
doi: 10.1523/JNEUROSCI.2793-06.2006.

DNA polymerase-beta is expressed early in neurons of Alzheimer's disease brain and is loaded into DNA replication forks in neurons challenged with beta-amyloid

Agata Copani  1 , Jeroen J M Hoozemans, Filippo Caraci, Marco Calafiore, Elise S Van Haastert, Robert Veerhuis, Annemieke J M Rozemuller, Eleonora Aronica, Maria Angela Sortino, Ferdinando Nicoletti
Affiliations
Comparative Study

DNA polymerase-beta is expressed early in neurons of Alzheimer's disease brain and is loaded into DNA replication forks in neurons challenged with beta-amyloid

Agata Copani et al. J Neurosci. .

Abstract

Cultured neurons exposed to synthetic beta-amyloid (Abeta) fragments reenter the cell cycle and initiate a pathway of DNA replication that involves the repair enzyme DNA polymerase-beta (DNA pol-beta) before undergoing apoptotic death. In this study, by performing coimmunoprecipitation experiments on cross-linked nucleoprotein fragments from Abeta-treated neurons, we demonstrate that DNA pol-beta coimmunoprecipitates with cell division cycle 45 (Cdc45) and with DNA primase in short nucleoprotein fragments. This indicates that DNA pol-beta is loaded into neuronal DNA replication forks after Abeta treatment. In response to Abeta the canonical DNA-synthesizing enzyme DNA pol-delta also was loaded into neuronal replication forks, but at later times than DNA pol-beta. Methoxyamine, an inhibitor of the apurinic/apyrimidinic endonuclease that allows for the recruitment of DNA pol-beta during the process of base excision repair (BER), failed to affect coimmunoprecipitation between DNA pol-beta and Cdc45, indicating that DNA pol-beta loading to the replication forks is independent of DNA breaks. However, methoxyamine reduced DNA replication and ensuing apoptosis in neurons exposed to Abeta, suggesting that an efficient BER process allows DNA replication to proceed up to the threshold for death. These data demonstrate that DNA pol-beta is an essential component of the DNA replication machinery in Abeta-treated neurons and additionally support the hypothesis of a close association of cell cycle events with neuronal death in Alzheimer's disease (AD). Accordingly, by investigating the neuronal expression of DNA pol-beta, along with phosphorylated retinoblastoma protein and neurofibrillary changes in AD brain, we show an early involvement of DNA pol-beta in the pathogenesis of AD.

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Figures

Figure 1.
Figure 1.
DNA pol-β loading at replication forks in Aβ-treated neurons. A, Representative immunoblots of DNA pol-β and DNA pol-δ in total protein extracts from cultured cortical neurons challenged with 25 μm Aβ25–35 for 4 or 8 h (Aβ, 4 h; Aβ, 8 h). B, Immunoprecipitation of 300 bp-sized nucleoprotein fragments with anti-primase antibody and blotting with Cdc45-specific antibody in controls (indicated as C) and Aβ-treated neurons. C, D, Also shown is immunoprecipitation of 1000 bp-sized nucleoprotein fragments with anti-Cdc45 antibody (C) or anti-primase antibody (D) and blotting with either DNA pol-β-specific or DNA pol-δ-specific antibodies. E, Representative immunoblot of APE-1 in 1000 bp-sized nucleoprotein fragments from neurons challenged with Aβ for 4 h both in the absence (–) and in the presence (+) of 15 mm methoxyamine (MTX). F, Immunoprecipitation of 1000 bp-sized nucleoprotein fragments with anti-Cdc45 antibody and blotting with either DNA pol-β-specific or DNA pol-δ-specific antibodies. Nucleoproteins were obtained from neurons challenged with Aβ for 4 or 8 h both in the absence (–) and in the presence (+) of 15 mm MTX.
Figure 2.
Figure 2.
Methoxyamine and low concentration hydrogen peroxide prevent Aβ-induced neuronal S phase and apoptosis. A, Protection by methoxyamine (MTX), aphidicolin (Aphi), and DDC against Aβ-induced apoptosis. Neurons were exposed to 25 μm Aβ25–35 for 18 h; S phase (A) and apoptosis (B) were scored by FACS analysis. Values are the means ± SEM of 10 determinations; *p < 0.05 [one-way ANOVA and Fisher's least significant difference (LSD)] versus Aβ alone. C, Protection by hydrogen peroxide (H2O2) against Aβ-induced degeneration. Neurons were exposed to 25 μm Aβ25–35 for 24 h. Values are expressed as a percentage of neuronal survival (quantified by MTT assay) and were calculated by four determinations; *p < 0.05 versus control (CTRL) and #p < 0.05 versus Aβ alone (one-way ANOVA and Fisher's LSD). In D and E, the neurons were exposed to 25 μm Aβ25–35 for 18 h, and both S phase (D) and apoptosis (E) were assessed by FACS analysis. Values are the means ± SEM of seven to nine determinations; *p < 0.05 versus control (CTRL) and #p < 0.05 versus Aβ alone (one-way ANOVA and Fisher's LSD).
Figure 3.
Figure 3.
DNA pol-β expression in human temporal cortex. A, DNA pol-β band (39 kDa) in samples of human testes and temporal cortex (Braak score III and VI) assayed in parallel by two different anti-DNA pol-β antibodies. B, Representative immunoblot of DNA pol-β in protein extracts of autoptic specimens from different Braak stages. The eEF2α band is shown as a loading control. C, Densitometer analysis of DNA pol-β expression levels from the indicated number of cases grouped according to the Braak score (0, I/II, III/IV, V/VI). Values are the means ± SEM of DNA pol-β normalized against eEF2α.
Figure 4.
Figure 4.
Immunohistochemical detection of DNA pol-β in control and AD temporal cortex. A, Human testis served as a positive control for the immunohistochemical detection of DNA pol-β. B, DNA pol-β immunoreactivity is observed in nuclei of the spermatids in the seminiferous epithelium. C, E, In control temporal cortex (Brodmann area 21), DNA pol-β immunoreactivity is observed in neurons (see insets). Immunoreactivity appears mainly in the nucleus of neurons in all layers (C, layer III/IV; E, layer V). Shown is a nondemented control case with Braak stage II and an immunoscore of 3. D, F, In AD temporal cortex DNA pol-β immunoreactivity is observed in only a few neurons (D, layer III/IV; F, layer V). Shown is an AD case with Braak stage VI and an immunoscore of 1. Scale bar: (in F) A, C–F, 160 μm; B, insets, 40 μm. G, Immunoreactive scores for DNA pol-β in the indicated number of cases grouped according to Braak's staging. Values are the means ± SEM of data obtained by semiquantitative scoring as follows: 0 = no staining, 0.5 = sporadic nuclear staining of neuronal cells, 1 = <10% nuclear staining, 2 = 10–50% nuclear staining, and 3 = >50% nuclear staining. *p = 0.001 versus Braak I/II (Dunn's test after rank-based ANOVA).
Figure 5.
Figure 5.
Immunohistochemical analysis of DNA pol-β, neurofibrillary changes, Aβ deposits, and ppRb. A, A case with only moderate neurofibrillary changes shows no strong association of nuclear DNA pol-β staining (brown) with AT8 immunoreactivity (red). B, A representative AD case with severe neurofibrillary changes. No immunoreactivity for DNA pol-β is observed after double immunolabeling. C, D, A nondemented control case with diffuse Aβ plaques (4G8; red) and nuclear immunostaining for DNA pol-β (brown). E, F, A case with strong immunoreactivity for DNA pol-β (E) and ppRb (F) shows a strong colocalization of these proteins in the nuclei of neuronal cells (G). Scale bar: (in G) A–C, 40 μm; D–G, 20 μm.
Figure 6.
Figure 6.
Correlation of DNA pol-β immunoreactivity with ppRb (top) and AT8 (bottom). All Braak stages (n = 31) were included. Scatter plots show the number of immunoreactive neurons scored as described in Figure 4G. Solid lines represent the best fit correlation. The positive correlation coefficient (r) indicates that DNA pol-β and ppRb immunoreactivities tend to increase together. The negative correlation coefficient indicates that DNA pol-β immunoreactivity tends to decrease while AT8 staining increases.
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