The FASEB Journal express article 10.1096/fj.02-0422fje. Published online October 18, 2002.

Erratic expression of DNA polymerases by β-amyloid causes neuronal death A. Copani,* M.A. Sortino,� A. Caricasole,� S. Chiechio,* M. Chisari,� G. Battaglia,¶ A.M. Giuffrida-Stella,§ C. Vancheri,|| and F. Nicoletti�,¶ Departments of *Pharmaceutical Sciences, �Experimental and Clinical Pharmacology, §Chemical Sciences, and ||Institute of Respiratory Diseases, University of Catania, Catania, 95125, Italy; �Department of Human Physiology and Pharmacology, University of Rome "La Sapienza", Rome, 00185, Italy; ¶I.N.M. Neuromed, Pozzilli, 86077, Italy. Corresponding author: Agata Copani, Department of Pharmaceutical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy. E-mail: acopani@katamail.com; caraless@ mbox.unict.it A. Copani and M.A. Sortino contributed equally to the work. ABSTRACT An ectopic reentrance into the cell cycle with ensuing DNA replication is required for neuronal apoptosis induced by β-amyloid. Here, we investigate the repertoire of DNA polymerases expressed in β-amyloid-treated neurons, and their specific role in DNA synthesis and apoptosis. We show that exposure of cultured cortical neurons to β-amyloid induces the expression of DNA polymerase-β, proliferating cell nuclear antigen, and the p49 and p58 subunits of DNA primase. Induction requires the activity of cyclin-dependent kinases. The knockdown of the p49 primase subunit prevents β-amyloid-induced neuronal DNA synthesis and apoptosis. Similar effects are observed by knocking down DNA polymerase-β or by using dideoxycytidine, a preferential inhibitor of this enzyme. Thus, the reparative enzyme DNA polymerase-β unexpectedly mediates a large component of de novo DNA synthesis and apoptotic death in neurons exposed to β-amyloid. These data indicate that DNA polymerases become death signals when erratically expressed by differentiated neurons. Key words: Alzheimer�s • cell cycle • DNA replication • apoptosis

R

ecent findings have highlighted the presence of markers of cell division in terminally differentiated neurons of the adult human brain. What is also intriguing is that these markers, such as cyclins and cyclin-dependent kinases (CDKs), are expressed by

degenerating neurons in Alzheimer�s disease (AD) and in a growing series of neurodegenerative disorders, including Down�s syndrome, frontotemporal dementia, progressive supranuclear palsy, and Niemann Pick�s disease (1). The question of whether expression of cell-cycle proteins truly drives the reactivation of a cell cycle has remained unsolved until the recent demonstration that DNA replication occurs in "at-risk" neurons of the AD brain (2) and in cultured cortical neurons challenged with β-amyloid peptide (βAP) (3). Cultured neurons exposed to full-length

βAP(1-42) or its active fragments βAP(1-40) or βAP(25-35) express the typical molecular repertoire necessary for the G1/S phase transition (3�5), enter the S phase, and die by apoptosis

(3), thus recapitulating the distinct features of degenerating neurons in the AD brain. Two fundamental questions should be answered for the understanding of how neurons degenerate in response to βAP: What specifically carries out DNA replication in differentiated neurons? and By which mechanism(s) does the ectopic S phase ultimately result in apoptotic death? In proliferating cells, de novo DNA synthesis is accomplished by the coordinated activity of DNA polymerases (DNA pol) α, -δ, and -ε, which follow one another in the elongation of the short RNA primers synthesized by DNA primase, the enzyme that forms an active complex with DNA pol-α (6). DNA pol-δ and/or -ε, which are chaperoned by the proliferating cell nuclear antigen (PCNA) (7), are also involved in several DNA repair events, including long-patch base excision repair (8) and mismatch repair (9). The single base excision repair pathway is served by DNA pol-β (10), which is not involved in replicative DNA synthesis likely because of its high level of infidelity (11). Present work investigates the repertoire of enzymes that allow neurons to start DNA replication in response to βAP and whether these enzymes have a role in βAP-induced apoptosis. MATERIALS AND METHODS Culture preparation and handling of βA peptides All experiments were performed in compliance with the European Union use of laboratory animals and the guidelines of the Italian D.L. 27/1/92 n. 116, art. 7, and according to the institutional approved protocol number 9213. Cultures of pure cortical neurons were obtained from E15 rat embryos according to a previously published method that yields >99% pure neuronal populations as assessed by immunofluorescent staining for neuron-specific MAP-2 (3). In brief, dissociated cortical cells were plated on 35-mm Nunc dishes precoated with 0.1 mg/ml poly-D-lysine at a density 2 × 106/dish and maintained in Dulbecco�s modified Eagle�s medium DMEM/Ham's F12 for 8�12 days in vitro (DIV). Cytosine-β-D-arabinofuranoside (10 µM) was added to the cultures 18 h after plating and kept for 4 days before medium replacement. βAP(25-35) or its reverse peptide βAP(35-25), purchased from Bachem Feinchemikalien (Bubendorf, Switzerland), were applied to mature neuronal cultures between 8 and 12 DIV. Different lots of peptides were used. Peptides were solubilized in sterile, doubly distilled water at an initial concentration of 2.5 mM, and aliquots were stored frozen at �20oC. βAP(25-35) and βAP(35-25) were used to a final concentration of 25 µM in the presence of the ionotropic glutamate receptor antagonists MK-801 (10 µM) and DNQX (30 µM) to avoid the participation of endogenous excitotoxic mechanisms (3). Addition of antisense oligonucleotides to the cultures The following �end-capped� phosphorothioate antisenses (obtained by MWG-Biotech [Florence, Italy]) were used: p49-primase (pr (p49)-As), 5'-tgaaggactggtagcgaatg-3'; pr-Sn, 5'-cattcgctaccagtccttca-3' (bases 170-189 of the rat mRNA sequence AJ011608); pol β-As, 5'-tacttgtggatcgcctggct-3'; pol β-Sn, 5'-agccaggcgatccacaagta-3' (bases 95-114 of the rat mRNA

sequence NM017141). Cultures were treated with oligonucleotides (1.5 µM) 16 h before the addition of βAP. FACS analysis for simultaneous assessment of S phase and apoptosis Neurons were processed for FACS analysis as described previously (3). In brief, neurons were harvested by incubation with 0.25% trypsin for 3 min and collected by low-speed centrifugation after the addition of 50% fetal calf serum. Neuronal pellets were fixed in 70% ethanol and treated for 1 h at 37oC with RNAse (100 µg/ml) before propidium iodide staining (50 µg/ml for 30 min). Therefore, samples were simultaneously analyzed for cell-cycle and apoptotic degeneration. DNA content and ploidy were assessed by using a Coulter Elite flow cytometer (Beckman Coulter, Milan, Italy), and cell-cycle distribution profiles were analyzed with the Multicycle AV software program. Apoptotic neurons were scored from the area of hypoploid DNA preceding the G0/G1 DNA peak. Immunoblotting Western blot analysis was carried out on total cell extracts as described previously (3). Electrophoresis was performed using 100 µg of cell proteins per lane. The following primary antibodies were used: anti-goat DNA pol-δ catalytic subunit (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), anti-mouse PCNA (2.5 µg/ml; Oncogene, Boston, MA), anti-mouse DNA pol-β (1:250; LabVision, Fremont, CA), anti-rabbit DNA pol-α/primase (1:200; kindly provided by Dr. David T.W. Wong and Dr. Takanori Tsuji, Harvard University [12]). The anti-mouse p53 antibody (5 µg/ml) was purchased from Oncogene. Specific hybridization signals were obtained by using horseradish peroxidase-conjugated secondary antibodies, followed by the SuperSignal West Pico chemiluminescence detection system (Pierce, Rockford, IL). Reverse transcriptase-polymer chain reaction (RT-PCR) analysis RT-PCR analysis was performed on total RNA, as described previously (13). PCR amplifications were carried out using the following pairs of primers: DNA pol-α (p180) Fw, 5'-CTGGCTGCCTTGGTGACATA-3'; DNA pol-α (p180) Rv, 5'-TCCGGTGTCCTTGGCAAGAT-3'; primase (p58) Fw, 5'-TTGCTGACGCTCTGGACCTG-3'; primase (p58) Rv, 5'-CCGGCCTCCATGACGAAGAT-3'; primase (p49) Fw, 5'-GTGTATTCAGGAAGGAGAGG-3'; primase (p49) Rv, 5'-GAACTGTCTCAGGAACAAGG-3'; DNA pol-β Fw, 5'-CAAGGACAGGAGTGAATGAC-3'; DNA pol-β Rv, 5'-AGTGACTTCTGGAGATGGCT-3'. For β-actin cDNA amplification, the primers were those described by Roelen et al. (14), which span an intron and yield products of different sizes (400 or 600 bp) depending on whether cDNA or genomic DNA is used as a template. RESULTS Cell cycle-dependent expression of DNA polymerases in βAP-treated neurons

We examined whether βAP induced changes in the expression of DNA polymerases in cultures of rat cortical neurons that are virtually devoid of astrocytes or other proliferating cells (3). Cortical

neurons constitutively expressed both DNA pol-δ and PCNA (Fig. 1a, CTRL), whereas DNA pol-ε was undetectable (data not shown). Upon exposure to 25 µM βAP(25-35), DNA pol-δ protein levels did not change, whereas PCNA levels substantially increased after 4 h (2.2 + 0.45 fold vs. controls, n=3; normalized by the levels of DNA pol-δ). PCNA levels remained high up to 16 h of exposure to βAP (Fig. 1a). To examine whether the induction of PCNA was related to cell-cycle activation, we treated the cultures with 300 nM of the CDK inhibitor flavopiridol (FLV) (15), which was protective against ßAP-induced apoptosis (% neuronal survival: control = 100 ± 3.3; 24 h βAP = 57.8 ± 4.4; 24 h ßAP ± FLV = 82.6 ± 4.3 [P<0.05 vs. βAP alone]; 24 h FLV = 95 ± 1.1) (also see ref 4). FLV reduced both constitutive and ßAP-induced PCNA levels, although it did not affect the constitutive expression of DNA pol-δ. Interestingly, FLV reduced the increased expression of PCNA after 8 and 16 h, but not after 4 h of exposure to βAP (Fig. 1a). Because FLV may inhibit protein kinases other than CDKs (16), we have also used mimosine as an alternative CDK inhibitor. Treatment of cultures with mimosine (400 µM), which prevents both βAP-induced S phase and apoptosis (3), reduced both basal and βAP-stimulated PCNA expression similarly to what observed with FLV (data not shown). We extended the study to DNA pol-β, which may be directed by PCNA in the long-patch DNA base-excision repair (17). DNA pol-β protein and mRNA levels were low in control cultures (Fig. 1a and 1c; CTRL) and were substantially increased following 2�16 h of exposure to βAP (Fig. 1a and 1c). The extent of increase in DNA pol-β protein, which was stable between 4 and 16 h, ranged from 1.7- to 3.5-fold in different experiments (n=6) due to a variability in the basal expression of the enzyme. As shown for PCNA, the cell-cycle inhibitors FLV (Fig. 1a) and mimosine (data not shown) decreased the induction of DNA pol-β after 8 or 16 h, but not after 4 h of exposure to βAP. Thus, as opposed to DNA pol-δ, DNA pol-β was induced by βAP, and, similar to PCNA, a late component in DNA pol-β induction required CDK activation. The cell-cycle-dependent induction of DNA pol-β, which in proliferating cells is always expressed at a constant low level (18), suggested that DNA pol-β was involved in an unusual type of DNA replication in βAP-treated neurons. Accordingly, βAP-treated neurons did not express the 180-kD catalytic subunit of the DNA pol-α/primase complex, although they did express the 49- and 58-kD primase subunits in a FLV-sensitive manner (Fig. 1b and 1c). Note that p58 primase and DNA pol-β mRNA levels remained stable or even increased with time of exposure to βAP (at least up to 16 h), whereas p49 primase mRNA levels were high after 2�8 h but then decreased after 16 h of exposure to βAP (Fig. 1c), perhaps as a result of a low stability of p49 mRNA. Irregularly expressed DNA polymerases accounted for DNA replication and apoptosis in βAP-treated cortical neurons We used antisense oligonucleotides (1.5 µM) to examine whether the induced expression of DNA pol-ß had any role in neuronal DNA replication and apoptosis. The antisense-induced knockdown of DNA pol-β significantly, but only partially, reduced the percentage of neurons entering S phase and undergoing apoptosis following exposure to βAP (Fig. 2a, 2b, 2d, and 2e).

Analogous effects were induced by the base analog dideoxycytidine (DDC, 100 µM), which preferentially inhibits DNA pol-β (19) (Fig. 2d and 2e). Treatment with antisenses directed against the p49 subunit of DNA primase also partially reduced both βAP-induced neuronal S phase and apoptosis (Fig. 2c�2e), according to the notion that primase activity is an obligatory requirement for de novo DNA synthesis. Treatment of the cultures with a p49 primase sense oligonucleotides also produced a small, but significant, reduction in βAP-induced S phase and apoptosis (see Fig. 2 and 2e). The nature of this effect, which was significantly smaller than that produced by p49 primase antisenses, is unclear. In contrast, the mixed DNA pol-α/δ inhibitor, aphidicolin (8 µg/ml) (20), reduced S phase but did not affect βAP-induced apoptosis (Fig. 2d and 2e). A causal role for an aphidicolin-insensitive DNA pol-β-directed replication in βAP-induced apoptosis was strengthened by the evidence that the knockdown of DNA pol-β reduced βAP-increased expression of the pro-apoptotic factor, p53 (Fig. 3a and 3b). DISCUSSION In pure neuronal cortical cultures, a reentrance into the cell cycle with ensuing DNA replication is required for βAP-induced apoptosis (3), suggesting that the molecular machinery carrying out DNA replication in neurons stays at the root of apoptotic degeneration. Present results show that overexpression of DNA pol-β is instrumental for the unscheduled DNA replication and neuronal death in response to βAP. DNA pol-β has been implicated in the base excision repair pathway (10); however, the biological significance of this enzyme is still uncertain. The known ability of DNA pol-β to fill DNA gaps must be essential in immature neurons, given that DNA pol-β-deficient mice undergo extensive apoptosis during neurogenesis (21). The repair activity of DNA pol-β seems to be equally relevant in the adult brain, because the age-dependent decline of the enzyme leads to defective DNA repair in aging neurons (22). Thus, the physiological role of DNA pol-β appears to be restricted to repair synthesis; however, evidence exists that DNA pol-β may participate in genome replication under some circumstances. DNA pol-β can substitute for DNA polymerase I in bacterial growth (23), and it can also carry out DNA endoreduplication in trophoblast cells (24). More recently, DNA pol-β has been found to compete with replicative DNA pols during replication in vitro of duplex DNA. In accordance with its properties to fill gapped DNA, DNA pol-β interferes with the synthesis of DNA lagging strand, which in fact requires the joining of Okazaki fragments (19). In addition, up-represented DNA pol-β lowers replication fidelity, thus increasing the mutagenicity rate of the system (19). Accordingly, the overexpression in mammalian cells of DNA pol-β has been found to increase the rate of spontaneous mutagenesis (25). These data support the hypothesis that an excess of the error-prone DNA pol-β can perturb the specific functions of DNA pol-δ and/or -ε during DNA replication and repair, thus affecting genomic stability in proliferating cells (26, 27). An up-regulation of DNA pol-β occurs in cancer cells (27, 28) perhaps in response to the carried load of DNA damage; otherwise, the levels of DNA pol-β are constant throughout the cell cycle of proliferating cells (18). βAP-treated neurons provide an unique example of a cell-cycle-dependent induction of DNA pol-β, indicative per se of an unusual type of DNA replication. The evidence that βAP-treated

neurons did not express the 180-kD catalytic subunit of the DNA pol-α/primase complex, but only the 58- and 49-kD primase subunits, strengthens this hypothesis. Hence, we hypothesize the existence of a non-canonical pathway of DNA synthesis that is mediated by DNA pol-β and is causally related to neuronal death in βAP-treated neurons. Given that DNA pol-β replicates DNA with a high infidelity and without adequate proofreading activity (11), the possibility has been raised that an increased expression of DNA pol-β generates DNA damage, eventually leading to cell death. Accordingly, increased DNA mutagenesis and apoptotic death in response to ionizing radiation (29) or oxidative damage (30) has been observed in recombinant cells overexpressing DNA pol-β. In neurons challenged with βAP, a pol-β-directed DNA replication might produce DNA damage, thus contributing to reach the threshold for the activation of a p53-dependent apoptotic pathway (31). The evidence that the increase in p53 expression was abolished in cultures treated with DNA pol-β antisenses is consistent with this hypothesis. A second pathway of DNA replication, which is aphidicolin-sensitive and is possibly mediated by DNA pol-δ, also occurred in response to βAP but was apparently unrelated to neuronal death. This does not exclude that components of the pathway preceding DNA elongation by pol-δ can signal death. Accordingly, an S-phase checkpoint dependent on primase activity but independent of DNA synthesis has been described (32). This might help explain the partial protective effect of DNA pol-β antisenses on neuronal apoptosis. Finally, note that besides DNA replication, DNA polymerases participate in checkpoint control systems through sophisticated protein-protein interactions (33). This high-order level of control might turn on the death pathway to correct the aberrant expression of replicative enzymes in neurons even independently of p53 activation. Accordingly, a p53-independent component of βAP-induced neuronal death has been described (5, 34). Taken collectively, these findings suggest that DNA polymerases become components of the death pathway in differentiated neurons, and this unsuspected role of DNA polymerases offers a novel explanation for the link of cell-cycle reactivation to apoptosis in AD. In addition, the induction of the primase subunits p49 and p58 in response to βAP provides the first evidence that proteins directly related to DNA replication can be reexpressed in stable postmitotic cells, such as neurons. Because reactivation of a quiescent cell-cycle machinery is a common theme in many neurodegenerative conditions (1), these findings might provide a novel mechanistic basis for neuronal death in human pathology. ACKNOWLEDGMENTS We thank Professor Maurizio Memo (University of Brescia, Italy) for his helpful suggestions and Dr. David T.W. Wong and Dr. Takanori Tsuji (Harvard University, Boston, MA) for kindly providing the DNA pol-α/primase antibody. We also thank Hoechst Marion Roussel (Tokio, Japan) for donating the flavopiridol. This work was supported by a grant from the Alzheimer�s Association (Chicago, IL).

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Received June 13, 2002; accepted September 2, 2002.

Fig. 1

Figure 1. Induction of DNA polymerases in cultured cortical neurons challenged with βAP(25-35) (25 µM) in the absence or presence of flavopiridol (FLV, 0.3 µM). a) Representative immunoblots of DNA pol-δ, PCNA, and DNA pol-β; b) induction of the p49 and p58 primase subunits of the DNA pol-α/primase complex. The right lane shows the expression in C6 glioma cells used as positive controls. Cultures were treated with βAP + FLV for the indicated times (a); cultures were treated with βAP + FLV for 8 h (b). No induction of DNA pol-β or PCNA was ever seen in cultures treated with the reverse peptide, ßAP(35-25) (25 µM, data not shown). c) RT-PCR analysis of mRNA for DNA pol-β, and for the p180, p49, and p58 subunits of the DNA pol-α/primase complex. Cultures were treated with βAP for the indicated times. Expression of mRNA in the rat testis is shown as a positive control. The 600-bp actin band, assessing genomic DNA contamination, was undetectable in all samples.

Fig. 2

Figure 2. Specific role of DNA polymerases in βAP-induced DNA replication and apoptosis in cortical neurons. Cultures were treated with the following antisense (As) or sense (Sn) end-capped phosphorothioate oligonucleotides: p49-primase (pr [p49]-As) and (pr [p49]-Sn); polβ-As and polβ-Sn. Cultures were treated with oligonucleotides (1.5 µM) 16 h before the addition of βAP. DDC (100 µM) and aphidicolin (Aphi, 8 µg/ml) were coapplied with βAP. RT-PCR of DNA pol-β mRNA (a), the immunoblot of DNA pol-β (b), and the RT-PCR of the p49 primase mRNA (c) in cultures treated with the respective As or Sn oligonucleotides; these were then exposed to βAP for 8 h (a, c) or 20 h (b). The effect of antisenses, aphidicolin, or DDC on the induction of S phase (d) and apoptosis (e) in cultures exposed to βAP for 18 h is shown. The 18-h time point has been selected to examine simultaneously βAP-induced S phase and apoptosis (3). Values are means ±SE of 11–20 determinations pooled from six independent experiments and are expressed as percent of βAP-induced neuronal S phase or apoptosis. In the six experiments, βAP induced S phase in 8.5–11.2% and apoptosis in 48–66% of the neuronal population (see ref 3 for an examination of the temporal relationship between S phase and apoptosis). P<0.05 (one-way ANOVA followed by Fisher’s LSD test) vs. *βAP alone or vs. #the respective Sn.

Fig. 3

Figure 3. DNA pol-β antisenses decrease βAP-induced p53 expression. a) Representative immunoblot of p53 in protein extracts from cortical neurons that have been treated with βAP for 24 h in the absence or presence of DNA pol-β antisenses (As) or senses (Sn). b) Densitometer scan analysis from three individual experiments. Values are expressed as percentages of controls (CTRL) and represent means ±SE *P<0.05 (one-way ANOVA followed by Fisher’s LSD test) vs. CTRL or βAP + polβ-As.