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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online October 18, 2002 as doi:10.1096/fj.02-0422fje. |
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,2
,¶
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; and
¶ I.N.M. Neuromed, Pozzilli, 86077, Italy
3Correspondence: Department of Pharmaceutical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy. E-mail: acopani@katamail.com; caraless{at}mbox.unict.it
SPECIFIC AIMS
Reactivation of a quiescent cell cycle machinery takes place in many neurodegenerative conditions, including Alzheimer’s disease (AD). Neurons of the AD brain undergo DNA replication, as do cortical neurons challenged with ß-amyloid peptide (ßAP). The evidence that DNA replication is required for neuronal apoptosis induced by ßAP suggests that the molecular machinery carrying out DNA replication in neurons is at the origin of apoptotic degeneration. We sought to investigate the repertoire of DNA polymerases expressed in ßAP-treated neurons and their specific role in DNA synthesis and apoptosis.
PRINCIPAL FINDINGS
1. Cell cycle-dependent expression of DNA polymerases in ßAP-treated neurons
To investigate the repertoire of enzymes that allow neurons to start DNA replication in response to ßAP, we examined whether ßAP-treated neurons express DNA polymerases, known to carry out DNA replication in proliferating cells. De novo DNA synthesis is accomplished by the coordinated activity of DNA polymerases (DNA pol) 
, -
, and -
, 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-.
Pure rat cortical neurons that constitutively expressed both DNA pol- and PCNA responded to 25 µM ßAP (25–35) with increased PCNA levels at 4–16 h and no change in DNA pol- protein levels (Fig. 1
a).
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The cyclin-dependent kinase (CDK) inhibitor flavopiridol (FLV, 300 nM), which was protective against ßAP-induced apoptosis [% neuronal survival: control = 100 ± 3.3; 24 h ßAP = 57.8 ± 4.4; 24 h ßAP + FLV = 82.6 ± 4.3*; 24 h FLV = 95 ± 1.1; *P < 0.05 vs. ßAP alone], reduced constitutive and ßAP-induced PCNA levels, but did not affect the constitutive expression of DNA pol-. FLV reduced the increased expression of PCNA after 8 and 16 h, but not after 4 h of exposure to ßAP (Fig. 1a
).
We extended the study to DNA pol-ß, the single base excision repair enzyme that may be directed by PCNA in the long-patch DNA base excision repair. DNA pol-ß protein and mRNA levels were low in control cultures and substantially increased after 2–16 h of exposure to ßAP (Fig. 1a, c
). FLV (Fig. 1a
) decreased the induction of DNA pol-ß after 8 or 16 h, but not after 4 h of exposure to ßAP. Thus, as opposed to DNA pol-, DNA pol-ß was induced by ßAP and, similar to PCNA, a late component in DNA pol-ß induction required CDK activation.
The unexpected cell cycle-dependent induction of DNA pol-ß suggested the existence of an unusual type of DNA replication in ßAP-treated neurons. Accordingly, ßAP-treated neurons did not express the 180 kDa catalytic subunit of the DNA pol-/primase complex, although they did express the 49 and 58 kDa primase subunits in an FLV-sensitive manner (Fig. 1b, c
).
2. Irregularly expressed DNA polymerases account for DNA replication and apoptosis in ßAP-treated cortical neurons
We used antisense oligonucleotides (1.5 µM) to examine whether induced expression of DNA pol-ß had any role in neuronal DNA replication and apoptosis. The antisense-induced knockdown of DNA pol-ß substantially reduced DNA synthesis and apoptotic death in neurons exposed to ßAP (Fig. 2
a, b, d, e). Analogous effects were induced by the base analog dideoxycytidine (DDC, 100 µM), which preferentially inhibits DNA pol-ß (Fig. 2d, e
).
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Treatment with antisense oligonucleotides directed against the p49 subunit of DNA primase reduced both ßAP-induced neuronal S phase and apoptosis (Fig. 2c-e
), according to the notion that primase activity is an obligatory requirement for de novo DNA synthesis.
In contrast, the mixed DNA pol-/ inhibitor aphidicolin (8 µg/mL) reduced DNA synthesis but did not affect ßAP-induced apoptosis (Fig. 2d, e
).
A causal role for an aphidicolin-insensitive DNA pol-ß-directed replication in ßAP-induced apoptosis was strengthened by evidence that the knockdown of DNA pol-ß reduced ßAP-increased expression of the proapoptotic factor p53.
CONCLUSIONS AND SIGNIFICANCE
Present results strengthen the hypothesis that ßAP kills neurons by activating an unscheduled mitotic cycle and show that overexpression of DNA pol-ß is instrumental for DNA replication and neuronal death in response to ßAP. The physiological role of DNA pol-ß appears restricted to DNA repair (Fig. 3
, left box); however, the biological significance of this enzyme is still uncertain.
|
ßAP-treated neurons provide a unique example of a cell cycle-dependent induction of DNA pol-ß, indicative per se of an unusual type of DNA replication. The evidence that ßAP-treated neurons do not express the 180 kDa catalytic subunit of the DNA pol-/primase complex, but only the 58 and 49 kDa primase subunits, reinforces this hypothesis. Hence, we propose the existence of a noncanonical pathway of DNA synthesis that is mediated by DNA pol-ß and is causally related to neuronal death in ßAP-treated neurons (Fig. 3
, right box).
The possibility has been raised that an increased expression of error-prone DNA pol-ß generates DNA damage eventually leading to cell death. In neurons challenged with ßAP, a pol-ß-directed DNA replication might produce DNA damage, thus contributing to reach the threshold for the activation of a p53-dependent apoptotic pathway (Fig. 3)
. The reduced p53 expression observed in cultures treated with DNA pol-ß antisense is consistent with this hypothesis.
Besides DNA replication, DNA polymerases participate in checkpoint control systems through sophisticated protein–protein interactions. This high-order level of control might turn on the death pathway to correct for the aberrant expression of replicative enzymes in neurons even independent of p53 activation (Fig. 3
, dashed arrow). Accordingly, a p53-independent component of ßAP-induced neuronal death has been described.
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. Induction of the primase subunits p49 and p58 in response to ßAP provides the first evidence that proteins directly related to DNA replication can be reexpressed in stable postmitotic cells such as neurons.
As reactivation of a quiescent cell cycle machinery is a common theme in many neurodegenerative conditions, these findings might provide a novel mechanistic basis for neuronal death in human pathology.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0422fje; to cite this article, use FASEB J. (October 18, 2002) 10.1096/fj.02-0422fje 
2 These authors contributed equally to the work.
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