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Molecular and Cellular Biology, November 2004, p. 9470-9477, Vol. 24, No. 21
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.21.9470-9477.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

p53 Deficiency Rescues Neuronal Apoptosis but Not Differentiation in DNA Polymerase ß-Deficient Mice

Noriyuki Sugo,1,{dagger} Naoko Niimi,1 Yasuaki Aratani,1 Keiko Takiguchi-Hayashi,2* and Hideki Koyama1*

Kihara Institute for Biological Research and Graduate School of Integrated Science, Yokohama City University, Totsuka-ku, Yokohama,1 Mitsubishi Kagaku Institute of Life Sciences, Machida-shi, Tokyo, Japan2

Received 27 February 2004/ Returned for modification 29 March 2004/ Accepted 5 August 2004


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In mammalian cells, DNA polymerase ß (Polß) functions in base excision repair. We have previously shown that Polß-deficient mice exhibit extensive neuronal cell death (apoptosis) in the developing nervous system and that the mice die immediately after birth. Here, we studied potential roles in the phenotype for p53, which has been implicated in DNA damage sensing, cell cycle arrest, and apoptosis. We generated Polß–/– p53–/– double-mutant mice and found that p53 deficiency dramatically rescued neuronal apoptosis associated with Polß deficiency, indicating that p53 mediates the apoptotic process in the nervous system. Importantly, proliferation and early differentiation of neuronal progenitors in Polß–/– p53–/– mice appeared normal, but their brains obviously displayed cytoarchitectural abnormalities; moreover, the mice, like Polß–/– p53+/+ mice, failed to survive after birth. Thus, we strongly suggest a crucial role for Polß in the differentiation of specific neuronal cell types.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Repair of DNA damage is essential for maintaining the integrity of the genetic information necessary for normal development and physiological consequences (28). DNA polymerase ß (Polß) is a 39-kDa protein of a single polypeptide, consisting of two catalytic, functional domains. The N-terminal 8-kDa domain carries a 5'-deoxyribose phosphate lyase activity, whereas the C-terminal 31-kDa domain carries a polymerase activity that fills a short gap with a 5'-phosphate (26, 40). Polß is a critical component of the base excision repair (BER) pathway. The BER pathway repairs DNA damage, such as apurinic/apyrimidinic (AP) sites and base modifications, which spontaneously occur or are induced by a variety of endogenous and exogenous agents, including reactive oxygen species and DNA alkylating agents. Biochemical studies have identified two types of BER in mammalian cells: a short-patch pathway involving replacement of one nucleotide and a long-patch pathway involving gap-filling of several nucleotides (46). The BER pathway is generally initiated by a specific DNA glycosylase that recognizes and removes a damaged base to generate an AP site in DNA, followed by incision of the site by an AP endonuclease. In the short-patch BER pathway, Polß removes the 5'-deoxyribose phosphate and fills the single nucleotide gap, and finally DNA ligase I or a complex of XRCC1 and DNA ligase III ligates the nick. On the other hand, the long-patch BER pathway requires proliferating cell nuclear antigen (PCNA), flap endonuclease 1 (FEN-1), and DNA ligase I to excise a flap-like structure resulting from strand displacement by Polß and/or Pol{delta}/{varepsilon} and to ligate the nick (6).

We and others previously showed that Polß-deficient mice exhibit a reduced size and weight and die with a respiratory defect immediately after birth (13, 42). In Polß-deficient mice, extensive cell death (apoptosis) occurs in postmitotic neurons in the developing central and peripheral nervous systems (42). This neuronal apoptosis is closely associated with the period between the onset and cessation of neurogenesis. Abnormalities in embryonic tissues other than the nervous system have not yet been reported (13, 19, 21). Therefore, we suggested that Polß plays an essential role specifically in the development of the nervous system (42). However, the cause of this neuronal apoptosis remains entirely unknown. Mouse embryonic fibroblast cells in culture, derived from a Polß knockout mouse, are viable and show normal growth characteristics (41). Although the mutant cells exhibit BER defects, as evidenced by increased sensitivity to DNA alkylating agents, their cell extracts still retain an activity to repair a damaged base residue in DNA substrate (6), indicating that there are both Polß-dependent and -independent BER pathways in vivo.

The tumor suppressor protein p53 plays a prominent role in the maintenance of genomic integrity (27). It is activated by different types of DNA damage, including single-strand breaks (SSBs), double-strand breaks (DSBs), and adducts, which are generated by endogenous or exogenous mechanisms. The activated p53 has a choice of cell cycle arrest for repair or apoptosis, depending on the level of damaged DNA; i.e., unless the damage is repaired, p53 leads to apoptosis. Recent studies show that p53 directly interacts with Polß, stimulating BER activity (37, 50). In the nervous system, it has been shown that p53 regulates neuronal apoptosis after neuronal injury induced by excitotoxins, hypoxia, and ischemia that cause oxidative damage (3, 29, 33, 47). In p53-deficient mice, kainic acid excitotoxicity- or ischemia-induced brain damage is significantly reduced (10, 34). These observations suggest the involvement of p53 in the control of neuronal apoptosis.

A close link between DNA damage and neurodegeneration appears evident from many pathological data and observations with mouse knockouts (8, 38). In mice deficient in DNA ligase IV (Lig4) and XRCC4, the main components of nonhomologous-end-joining apparatus for DSB repair, differentiating neurons undergo massive cell death (4, 14, 17, 22). However, this apoptosis is completely rescued by p53 deficiency (15, 16). It would be important to examine whether neuronal cell death found in Polß-deficient mice (42) is mediated by p53 activation. Here we study a potential role for p53 in the phenotypes, including neuronal cell death associated with Polß deficiency. We found that p53 deficiency rescues the neuronal apoptosis in a Polß-deficient background. However, it should be noticeable that Polß–/– p53–/– mice still exhibit cytoarchitectural defects in the development of the nervous system and die shortly after birth. These observations strongly suggest that Polß is crucial for the differentiation process of specific neuronal cell types.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice. Polß-deficient mice were described previously (42). p53-deficient mice (C57BL/6J-Trp53 tm1Tyj) were obtained from The Jackson Laboratory (24). PCR genotyping protocol for p53 targeted allele are directed on the website of JAX MICE, The Jackson Laboratory (http://jaxmice.jax.org). Noon of the day on which the vaginal plug was detected in the morning was designated embryonic day 0.5 (E0.5). All mice were maintained in a pathogen-free environment under the guidelines of Kihara Institute for Biological Research, Yokohama City University, for laboratory animals.

Histology, immunohistochemistry, and TUNEL assay. Embryos were perfused with 4% paraformaldehyde and 7% picric acid in 0.1 M sodium phosphate buffer (pH 7.4); the brain was removed and postfixed in the same fixative for 2 h, equilibrated with 25% sucrose-phosphate-buffered saline, frozen in OCT compound (Sakura Finetechnical Co.) and sectioned on a cryostat (10 µm). The sections were incubated with rabbit anti-p53 polyclonal antibody CM-5 (Novocastra Laboratories, 1:3,000), rabbit anti-cleaved caspase-3 polyclonal antibody (Cell Signaling Technology; 1:100), mouse anti-PCNA monoclonal antibody PC10 (Sigma; 1:100), mouse anti-neuron specific type-III ß-tubulin monoclonal antibody Tuj1 (BabCO; 1:1,000), mouse anti-phosphorylated neurofilament SMI31 (Sternberger Monoclonal Antibodies; 1:4,000), and rabbit anti-calbindin/spot 35 polyclonal antibody (a kind gift of T. Yamakuni) (1). Cy3 or horseradish peroxidase-conjugated antibody was used for a secondary antibody to visualize primary antibody. TSA Biotin System (Perkin-Elmer Life Sciences) was applied to anti-p53, anti-calbindin immunohistochemistry. The TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay was performed on cryosections by using 0.12 U of TdTase (Roche)/µl with 0.5 µM biotin-14-dATP (Invitrogen) in 1x TdT buffer (Roche) with 1.5 mM CoCl2. Horseradish peroxidase-conjugated biotin (Jackson Immunoresearch Laboratories) was used for signal detection. Cell nuclei were stained with DAPI (4',6'-diamidino-2-phenylindole) for immunofluorescence. Cresyl violet (Sigma) staining was performed to show neuronal architecture.

Western blot analysis. Cell extracts were prepared from developing telencephalons in E13.5 embryos, electrophoresed in an 8.0% sodium dodecyl sulfate-polyacrylamide gel, and transferred to an Immobilon membrane (Millipore) as described previously (42). The membrane was probed with anti-human phospho-p53 (Ser-15) antibody (Cell Signaling Technology; 1:1,000) and peroxidase-conjugated goat anti-rabbit immunoglobulin G (Chemicon) and detected with enhanced chemiluminescent detection reagents (ECL Plus kit; Amersham).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Polß deficiency activates the p53-dependent apoptosis pathway. p53 is activated and stabilized after excessive DNA damage generated by endogenous or exogenous mechanisms, and induces apoptosis (27). We examined whether neuronal cell death found in Polß-deficient mice (42) was induced by such p53 activation. To detect DNA fragmentation in cells undergoing apoptosis, we performed terminal deoxynucleotidyltransferase-mediated dUTP biotin nick-end labeling (TUNEL) assay. In Polß+/+ (wild-type) mice, TUNEL-positive cells were not detected in developing neocortex at E13.5 (Fig. 1A). In contrast, in Polß–/– developing neocortex, an extensive number of TUNEL-positive cells were observed in the primordial plexiform layer (PPL) and intermediate zone (IZ), where postmitotic neuronal cells were present; but these cells were less detectable in the ventricular zone (VZ), where proliferating neuronal progenitor cells were present (Fig. 1D).



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  		FIG. 1. Polß deficiency induces apoptosis and activates p53 in the developing nervous system. Coronal sections of E13.5 developing neocortices in Polß+/+ (wild-type) (A to C) and Polß–/– (D to F) embryos were assayed by TUNEL staining (A and D) or immunohistochemistry with anti-cleaved caspase-3 antibody (B and E) and anti-p53 antibody (C and F). LV, lateral ventricle.

 
We next examined the expression of cleaved caspase-3 and p53 by immunohistochemistry in E13.5 developing neocortex. Since caspase-3 is activated by proteolytic cleavage of its inactive zymogen by apoptosis (35), cleaved caspase-3 is a useful marker for apoptotic cells. Anti-cleaved caspase-3 antibody could obviously detect apoptotic neuronal cells in Polß–/– embryos (Fig. 1E), as observed by the above TUNEL assay, whereas wild-type embryos were not stained (Fig. 1B). Importantly, a larger number of cells with higher p53 levels were seen in Polß–/– embryos than in wild-type (Fig. 1C versus 1F); in the VZ, such p53-stained cells were more frequent than the TUNEL- or cleaved caspase-3-positive cells. It is known that serine-15 of human p53 (equivalent to serine-18 in mouse p53) is phosphorylated in response to DNA damage (39). Therefore, we performed Western blot analysis with anti-phosphorylated serine-15 of human p53 specific antibody with cell extracts from E13.5 developing telencephalons of Polß+/+ p53+/+ (wild-type), Polß–/– p53+/+, and Polß–/– p53–/– embryos (generation of the double-mutant mice will be cited below). We found that the serine-18 of p53 in Polß–/– p53+/+ extracts was strongly phosphorylated compared to that in wild-type control, with no staining in Polß–/– p53–/– extracts (Fig. 2). These results suggest an intriguing possibility that neuronal apoptosis observed in Polß-deficient embryos is induced by p53 activation in response to DNA damage, immediately after final mitosis of the progenitor cells.



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  		FIG. 2. Western blot analysis of phosphorylated serine-18 of p53 with cell extracts prepared from E13.5 developing telencephalons of Polß+/+ p53+/+ (wild-type), Polß–/– p53+/+, and Polß–/– p53–/– embryos. The same amounts of protein were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting with anti-human phospho-p53 (Ser-15) antibody.

 
To explore potential physiological interactions between Polß and p53, we produced mice null for both Polß and p53. We bred Polß+/– and p53+/– mice and generated Polß+/– p53+/– double-heterozygous mice, which exhibited normal development and fertility. When Polß+/– p53+/– mice were intercrossed, developing embryos with nearly the expected Mendelian ratios of all genotypes were observed at E11.5 to E18.5 (Table 1). However, no offspring with Polß–/– p53+/+, Polß–/– p53+/–, and Polß–/– p53–/– genotypes was detected at weaning (ca. 4 weeks after birth). It should be noted that, like Polß–/– p53+/+ neonates, all Polß–/– p53+/– and Polß–/– p53–/– neonates died at postnatal day 1 (data not shown). With anti-cleaved caspase-3 antibody, we examined neuronal apoptotic phenotypes by staining sections of the developing telencephalon in E13.5 embryos and the spinal cord and dorsal root ganglion in E11.5 embryos of wild-type controls and littermates. In tissues of Polß–/– 53+/+ mice, a large number of cleaved caspase-3-positive neuronal cells were observed compared to those of control mice (compare Fig. 3A and E with 3B and F). However, these stained cells were dramatically decreased in Polß–/– p53+/– mice (Fig. 3C and G) and, more importantly, completely disappeared in Polß–/– p53–/– double-mutant mice (Fig. 3D and H). These observations indicate that p53 deficiency rescues the neuronal apoptosis associated with Polß deficiency and that p53 haploinsufficiency also substantially does so. These results indicate that the neuronal apoptosis in Polß-deficient mice is mediated by the p53-dependent apoptosis pathway.


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  		TABLE 1. Genotypic analysis of Polß+/– p53+/– intercrosses

 


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  		FIG. 3. Neuronal apoptosis in Polß-deficient mice is mediated by the p53-dependent pathway. Coronal sections of E13.5 telencephalons (A to D) and E11.5 spinal cords and dorsal root ganglions (E to H) in Polß+/+ p53+/+ (wild-type) (A and E), Polß–/– p53+/+ (B and F), Polß–/– p53+/– (C and G), and Polß–/– p53–/– (D and H) embryos were stained with anti-cleaved caspase-3 antibody. DRG, dorsal root ganglion; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; NCX, neocortex; SC, spinal cord.

 
Proliferation and early differentiation of progenitors during neurogenesis appear normal in E13.5 Polß–/– p53–/– mice. Proliferation and differentiation of progenitors are temporally and spatially controlled during neurogenesis (12). The generation of neurons from the progenitors involves successive steps in commitment and differentiation, which are progressively generating more restricted cell types. These steps include (i) cell type specification, (ii) exit from the cell cycle, (iii) differentiation into distinct cell types, (iv) migration into a correct destination, and (v) production of correct cell-cell contacts through dendritic and axonal processes. We examined effects of p53 deficiency on histogenesis of the neocortex by cresyl violet staining (Fig. 4A to F and 4P to R). In E13.5 wild-type lateral regions of the neocortex, generation of the PPL, IZ, and cortical plate (CP) was clearly observed (Fig. 4A), but the CP in the dorsal region was not (Fig. 4D). The ventrolateral-to-dorsomedial, morphological gradients are evident in the development of the neocortex (5). In contrast, in Polß–/– p53+/+ neocortices, the CP was not clearly seen due to an extraordinary number of both pyknotic and cleaved caspase 3-positive cells undergoing apoptosis (Fig. 4B, E, and H). However, in Polß–/– p53–/– neocortices, the CP was almost normally generated (Fig. 4C), parallel with a striking disappearance of apoptotic cells (Fig. 4F and I). With the use of antibody against either PCNA, a proliferating cell marker (Fig. 4J to L), or neuron-specific type III ß-tubulin, an early neuron marker (Fig. 4M to O), we observed the state of neuronal cells escaping from apoptosis in E13.5 developing neocortex. In the PPL and IZ of Polß–/– p53+/+ mice, expression of PCNA appeared in some apoptotic cells (Fig. 4K), whereas expression of type III ß-tubulin was reduced with increasing apoptotic cells (compare Fig. 4N and M). Expression of PCNA is regulated by p53 in response to ionizing radiation in neuronal cells (45). Therefore, the PCNA activation in the apoptotic cells of Polß–/– p53+/+ mice (Fig. 4K) might be due to a response to certain DNA damage. On the other hand, in Polß–/– p53–/– mice, we found intact expression of both PCNA and type III ß-tubulin, similar to wild-type controls (Fig. 4L and O, respectively). Neuronal progenitors expressing PCNA were restricted in the VZ (Fig. 4L); the differentiating neurons present in the PPL and IZ expressed type III ß-tubulin at a normal level (Fig. 4O). Finally, at E14.5, formation of the CP in Polß–/– p53–/– mice was more clearly observed than that in Polß–/– p53+/+ mice (compare Fig. 4R and Q). Together, it appears that in Polß–/– p53–/– embryos early differentiation of neuronal cells escaping from apoptosis proceeds normally.



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  		FIG. 4. Neuronal progenitors in E13.5 Polß–/– p53–/– embryos appear to normally proliferate and differentiate. Coronal sections of telencephalons in Polß+/+ p53+/+ (wild-type) (A, D, G, J, M, and P), Polß–/–p53+/+ (B, E, H, K, N, and Q), and Polß–/– p53–/– (C, F, I, L, O, and R) embryos at E13.5 (A to O) and E14.5 (P to R) were stained with cresyl violet (A to F and P to R). The sections were also stained with anti-cleaved caspase-3 antibody (G to I), anti-PCNA antibody (red) and DAPI (blue) (J to L), or anti-neuron specific type III ß-tubulin antibody TuJ1 (M to O). Arrows in panels A, C, P, Q, and R indicate the CP. Arrowheads in panel K indicate PCNA-activated cells. LV, lateral ventricle.

 
Formation of the nervous system is incomplete in E18.5 Polß–/– p53–/– mice. p53 deficiency dramatically rescued neuronal apoptosis in Polß-deficient mice (Fig. 3). As mentioned above, early neuronal differentiation appeared to proceed normally in E13.5 Polß–/– p53–/– embryos (Fig. 4). As shown in Table 1, Polß–/– p53–/– embryos could survive during gestation and died at postnatal day 1. Therefore, we examined the development of their brains at E18.5 by immunohistochemical analysis (Fig. 5). We observed slight but significant, neuronal defects in the telencephalon of Polß–/– p53–/– embryos. Cresyl violet staining revealed that, compared to Polß–/– p53+/+ mice, the size of the telencephalon and its cytoarchitecture were moderately recovered in Polß–/– p53–/– mice (compare Fig. 5B and C). We analyzed the axonal tract formation by staining phosphorylated neurofilaments. In Polß–/– p53+/+ brains, the major axonal tract, anterior commissure did not cross at the midline (Fig. 5E); notably, this defect could not be rescued by p53 deficiency (Fig. 5F). We also observed more aberrant axonal tracts in the striatum of Polß–/– p53+/+ brains (Fig. 5H) than in that of wild-type controls (Fig. 5G), and similar aberrations were observed in Polß–/– p53–/– brains (Fig. 5I). These results indicate that, at least in some areas, the brain development in Polß–/– p53–/– embryos was not complete. Recently identified is a cell type migrating from the lateral ganglionic eminence and the medial ganglionic eminence of the basal ganglia to the neocortex of telencephalons (2, 32). The tangentially migrating cells become interneurons synthesizing inhibitory neurotransmitter {gamma}-aminobutyric acid (GABA) in the CP of the neocortex. A great number of neuronal apoptotic cells were observed in both the lateral ganglionic eminence and the medial ganglionic eminence in Polß–/– p53+/+ embryos at E13.5 (compare Fig. 3A and B). We therefore examined whether GABAergic interneurons existed in the E18.5 neocortex. The interneurons in developing neocortex are known to express calbindin D28k (Calbindin), an intracellular calcium-binding protein (2, 32). In Polß–/– p53+/+ embryos (Fig. 5K), we found a significant decrease in the number of calbindin-positive cells in the CP compared to wild-type embryos (Fig. 5J). More importantly, this decrease was not rescued by p53 deficiency in Polß–/– p53–/– embryos (Fig. 5L). Taken together, these data, shown in Fig. 5, indicate that p53 deficiency does not completely rescue developmental defects in the central nervous system, associated with Polß deficiency. These phenotypes are in sharp contrast to those of Lig4–/– p53–/– and XRCC4–/– p53–/– mice, which can be alive for several weeks after birth. These results suggest a crucial role for Polß in the formation of the intact neuronal circuit and migration of certain neuronal cell types.



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  		FIG. 5. Development of the brain is incomplete in E18.5 Polß–/– p53–/– embryos. Coronal sections of telencephalons in Polß+/+ p53+/+ (wild-type) (A, D, G, and J), Polß–/– p53+/+ (B, E, H, and K), and Polß–/–p53–/– (C, F, I, and L) embryos at E18.5 were stained with cresyl violet (A to C), anti-phosphorylated neurofilament antibody SMI31 (D to I), and anticalbindin antibody (J to L). Photographs of the sections in each genotype were taken with the same magnification. The arrowheads in panels A, B, and C indicate part of the cytoarchitecture recovered moderately in Polß–/– p53–/– mice compared to Polß–/– p53+/+. The arrows in panels D, E, and F indicate the anterior commissure (AC). The arrows in panels G, H, and I indicate aberrant axonal tracts in the striatum (St). The arrows in panel J indicate calbindin-positive cells. MZ, marginal zone.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have shown here that p53 deficiency rescues neuronal apoptosis in Polß-deficient mice and that haploinsufficiency substantially does so as well (Fig. 3). We have also shown that p53 is activated by its serine-18 phosphorylation in developing telencephalons of Polß-deficient mice (Fig. 2). These results indicate that the neuronal apoptosis associated with Polß deficiency is mediated by the p53-dependent pathway. In addition, it should be noted that, like Polß–/– p53+/+ neonates, Polß–/– p53–/– neonates die shortly after birth.

The onset of p53-dependent repair or apoptosis is determined by the level of accumulated damaged DNA (36). There is ample biochemical evidence for functioning of Polß mainly in the short-patch BER pathway to repair SSBs, which are mostly generated as intermediates of damaged bases metabolized by DNA glycosylase and AP endonuclease (46). Mouse embryonic fibroblasts defective in Polß are highly sensitive to DNA alkylating agents but not to X-ray radiation (41, 42), supporting in vivo that Polß deficiency leads to defects in SSB repair but not in DSB repair. Thus, Polß deficiency should result in increased levels of SSBs, even if the Polß-independent long-patch BER is able to partially substitute for the short-patch BER. The increased SSB levels would stabilize and activate p53, leading to apoptosis during neuronal differentiation in Polß–/– mice. In mice defective in Lig4, XRCC4, Ku70/80, or XRCC2, which all function in DSB repair, differentiating neurons undergo massive apoptosis (4, 11, 14, 17, 22). Therefore, in these mice, unrepaired DSBs are thought to be the cause of the apoptosis (4, 11, 14, 17, 22). The apoptosis in Lig4–/– and XRCC4–/– embryos is rescued by p53 deficiency (15, 16). Lig4–/– p53–/– and XRCC4–/– p53–/– neonates can survive several weeks after birth without behavioral or neurological abnormalities. This is in sharp contrast with our observation that Polß–/– p53–/– neonates die shortly after birth. The degree and quality of rescue by p53 deficiency in repair-deficient mice appear to vary depending on the type and level of DNA damage. As discussed above, in Polß–/– mice, the damage is most likely SSBs, but the possibility that these SSBs are subsequently converted into DSBs in the final DNA replication of neuronal progenitor cells cannot be ruled out.

In Polß–/– p53–/– mice at E13.5, early steps of neuronal differentiation seem to proceed normally, as judged by immunohistochemical analysis (Fig. 4). However, at E18.5, these and Polß–/– p53+/+ mice displayed serious cytoarchitectural defects in the major axonal tract (Fig. 5E and F) (with more aberrant axonal tracts in the striatum [Fig. 5H and I]) and the migration in GABAergic interneurons (Fig. 5K and L). These results suggest that, although p53 deficiency indeed rescues neuronal apoptosis, these neurons are still incomplete as mature ones, implying that the deficiency cannot fully restore the neuronal development of at least certain cell types. The brain is composed of remarkably complex neuronal cell types and networks. In the development of the brain, cell migration, axon growth, and pathfinding are fundamental processes (12). Recent studies with knockout mice have identified a number of molecules responsible for such processes (30, 32). Loss of these molecules severely affects the brain development and is critical for survival. The abnormal development of the nervous system observed in both Polß–/– p53–/– and Polß–/– p53+/+ mice at E18.5 may be responsible for death shortly after birth. In Lig4–/– p53–/– or XRCC4–/– p53–/– mice, severe defects in lymphogenesis are never recovered by p53 deficiency, implying that Lig4 or XRCC4 is a critical factor for lymphogenesis (15, 16). Similarly, our finding that the neuronal differentiation in Polß–/– mice is not completely rescued by p53 deficiency strongly suggests that Polß is a critical factor for neurogenesis; that is, Polß may absolutely be required for neuronal differentiation.

The reason why Polß is required for neuronal differentiation remains obscure. One possibility is that in neuronal differentiation, a large amount of damaged bases and SSBs are generated by reactive oxygen species, which might occur particularly in some neuronal cell types actively undergoing migration and/or axon pathfinding. Recently, the Polß-dependent pathway was shown to be induced in response to oxidative base damage (7). Polß might specifically be required to repair those damaged bases and SSBs. Thus, Polß deficiency would lead to increased levels of DNA damage and activation of p53, eventually resulting in apoptosis. A second possibility is that Polß is involved in chromatin remodeling and transcription in neuronal differentiation. When neuronal progenitor cells become postmitotic neurons, they exit cell cycle and drastically alter the pattern of gene expression from immature to mature neurons (12). Transcriptional activation of a gene involves recruitment of not only a sequence-specific DNA-binding protein but also a coactivator complex, including proteins with chromatin-modifying activity. For example, DNA topoisomerase IIß alters DNA topology and forms complexes with proteins involved in chromatin remodeling and transcription (25, 44). The enzyme-deficient mice show defects in the laminar organization of the neocortex and motor axon growth, resulting in a breathing impairment and death of the pups shortly after birth (31, 49). This finding suggests that the control of chromatin reorganization is indispensable for neuronal differentiation. Interestingly, we note that transcriptional coactivator p300 forms a physical and functional interaction with Polß (23). p300 integrates a diverse signaling pathway for a number of sequence-specific transcription factors and activates transcription through chromatin remodeling via intrinsic histone acetyltransferase activity (20). Therefore, in association with p300 or related proteins, Polß might function to maintain the integrity of genes being, or to be, expressed in certain neuronal cell types. A third possibility is that during neuronal differentiation, a genomic rearrangement factor(s) is expressed and generates a certain type of DNA damage (repairable by Polß) to initiate a specific differentiation. In the immune system, the molecular mechanism of diversity by rearrangement of the immunoglobulin or T-cell receptor gene clusters is well understood (43). In V(D)J recombination, the lymphocyte-specific endonucleases RAG1 and RAG2 initially cleave specific recognition sequences in immunoglobulin loci, followed by completion of rearrangements through DSB repair by the action of nonhomologous-end-joining factors (18). Similarly, in the nervous system, neuronal diversity might be created by such genomic rearrangement (9, 48). If this is the case, DNA repair by Polß would be an essential part of the diversity mechanism.

In conclusion, our studies show that p53 deficiency dramatically rescues neuronal apoptosis associated with Polß deficiency, indicating that p53 mediates the apoptotic process in the nervous system. However, p53 deficiency cannot restore complete differentiation of neuronal progenitors and leads to lethality shortly after birth. These observations suggest a crucial role for Polß in differentiation of specific neuronal cell types. In addition, it is evident that in neuronal differentiation, p53 acts as a gatekeeper to maintain genomic stability against various types of DNA damage (27). Further studies will be needed to elucidate the precise role of Polß in neurogenesis.


   ACKNOWLEDGMENTS
 
We thank T. Yamakuni (Tohoku University) for the gift of the anti-calbindin antibody, Y. Tanabe (Mitsubishi Kagaku Institute of Life Sciences) for helpful discussion, and N. Adachi (Yokohama City University) for critical reading of the manuscript. We also thank C. Nishigaki for technical support and F. Oonuma for animal care.

N.S. is a recipient of Research Fellowship of the Japan Society for the Promotion of Science for Young Scientists. This study was supported in part by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.


   FOOTNOTES
 
* Corresponding author. Mailing address for Hideki Koyama: Kihara Institute for Biological Research and Graduate School of Integrated Science, Yokohama City University, 641-12 Maioka-cho, Totsuka-ku, Yokohama 244-0813, Japan. Phone: 81-45-820-1907. Fax: 81-45-820-1901. E-mail: koyama@yokohama-cu.ac.jp. Mailing address for Keiko Takiguchi-Hayashi: Research Laboratory 1 (CNS), Pharmaceuticals Research Unit, Research and Development Division, Mitsubishi Pharma Corp., 1000, Kamoshida-cho, Aoba-ku, Yokohama 227-0033, Japan. Phone: 81-45-963-3413. Fax: 81-45-963-3967. E-mail: Hayashi.Keiko{at}mp.m-pharma.co.jp. Back

{dagger} Present address: Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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Molecular and Cellular Biology, November 2004, p. 9470-9477, Vol. 24, No. 21
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.21.9470-9477.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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