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Comparative Study
, 26 (11), 2732-42

BRCA2 Is Required for Neurogenesis and Suppression of Medulloblastoma

Affiliations
Comparative Study

BRCA2 Is Required for Neurogenesis and Suppression of Medulloblastoma

Pierre-Olivier Frappart et al. EMBO J.

Abstract

Defective DNA damage responses in the nervous system can result in neurodegeneration or tumorigenesis. Despite the importance of DNA damage signalling, the neural function of many critical DNA repair factors is unclear. BRCA2 is necessary for homologous recombination repair of DNA and the prevention of diseases including Fanconi Anemia and cancer. We determined the role of BRCA2 during brain development by inactivating murine Brca2 throughout neural tissues. In striking contrast to early embryonic lethality after germ-line inactivation, Brca2(LoxP/LoxP);Nestin-cre mice were viable. However, Brca2 loss profoundly affected neurogenesis, particularly during embryonic and postnatal neural development. These neurological defects arose from DNA damage as Brca2(LoxP/LoxP);Nestin-cre mice showed extensive gammaH2AX in neural tissue and p53 deficiency restored brain histology but lead to rapid formation of medulloblastoma brain tumors. In contrast, loss of the Atm kinase did not markedly attenuate apoptosis after Brca2 loss, but did partially restore cerebellar morphology, supporting a genomic surveillance function for ATM during neurogenesis. These data illustrate the importance of Brca2 during nervous system development and underscore the tissue-specific requirements for DNA repair factors.

Figures

Figure 1
Figure 1
Brca2 loss leads to neurogenesis defects in a p53-dependent manner. (A) The Brca2LoxPLoxP;Nestin-cre (Brca2Nes-cre) mutant brain is substantially smaller than Brca2+/+;Nestin-cre (Brca2Ctrl) controls, although general morphology is intact. The cortex and the hippocampus (hippo) maintain relatively normal laminar structure (arrows). The cerebellum is markedly smaller in the mutant. (B) Reduced brain size at P21 in Brca2Nes-cre mice compared with that in Brca2Ctrl mice. Rescue of cerebella size occurs in the Brca2Nes-cre;p53−/− mice. (C) The relative brain weight of P7 Brca2Nes-cre and Brca2Nes-cre;p53−/− compared with Brca2+/+;Nes-cre (Brca2Ctrl) shows rescue by p53 loss. (D) H&E staining of Brca2LoxP/LoxP (Brca2Ctrl), Brca2Nes-cre and Brca2Nes-cre;p53−/− brain sections at P21 reveals size reduction of the mutant cerebellum compared with WT (magnification × 2). Calbindin (D-28K) staining shows the Purkinje cell layer is intact in the Brca2Nes-cre cerebellum (magnification × 40), although ectopic localization of Purkinje cells occurs in some lobules (E, magnification × 20). ML, molecular layer; PC, Purkinje cells; IGL, inner granular layer.
Figure 2
Figure 2
Brca2 loss leads to DNA damage and increased apoptosis in EGL granule cell progenitors. (A) Loss of Brca2 leads to H2AX phosphorylation (γH2AX) in Brca2Nes-cre and Brca2Nes-cre;p53−/− but not control; Brca2Ctrl (Brca2Flox/Flox) P7 cerebellum (magnification × 20); insets show higher magnification of γH2AX (magnification × 40). (B) Compared with control Brca2Ctrl (Brca2+/+;Nestin-cre) P7 cerebellum, apoptosis is widespread throughout the Brca2Nes-cre cerebellum, but is attenuated after associated loss of p53, as assessed by TUNEL or immunostaining for apoptosis-related single-stranded DNA (ssDNA) (magnification × 40). Quantification of TUNEL staining (C) or pyknotic nuclei (D) in Brca2+/+;Nes-cre (Brca2Ctrl) P7 EGL compared with Brca2Nes-cre showed a significant increase in apoptosis (P<0.0001), while apoptosis was attenuated in Brca2Nes-cre;p53−/− tissue. (E) Apoptotic cells with typical nuclear morphology of pyknosis commonly show colocalization with biochemical apoptotic markers (ssDNA immunostaining) (magnification × 100).
Figure 3
Figure 3
Apoptosis and proliferation analysis of Brca2Nes-cre cerebella. (A) Analysis of proliferation was determined after BrdU incorporation in control tissue and Brca2Nes-cre;p53+/+ and Brca2Nes-cre;p53−/− EGL. Proliferation is not perturbed by Brca2 loss, as BrdU incorporation is similar between Brca2Nes-cre and Brca2Ctrl. (B) Increased phosphorylated histone H3-positive cells were found in the Brca2Nes-cre and Brca2Nes-cre;p53−/− EGL (P<0.0001). Asterisks indicate significant differences and n indicates the number of different cerebella analyzed from each genotype.
Figure 4
Figure 4
Developmental analysis of Brca2 loss. Proliferation and apoptosis were determined at various developmental times in Brca2Ctrl and Brca2Nes-cre neural tissue. Analysis of apoptosis was performed using TUNEL and proliferation was determined after analysis of BrdU incorporation. (A) No difference in proliferation was found at early developmental times for the cerebellum. (B) Significantly increased apoptosis (P<0.0001) was found throughout neural development in mutant (Brca2Nes-cre) tissue; Brca2Ctrl was Brca2+/+;Nestin-cre. (C) Representative BrdU staining in the developing E14.5 hindbrain ventricular zone (magnification × 40). (D) Representative TUNEL staining in the developing E14.5 hindbrain ventricular zone (magnification × 40).
Figure 5
Figure 5
Atm inactivation restores cerebellar growth but not microcephaly in Brca2Nes-cre mice. (A) Partial rescue of cerebellar development in Brca2Nes-cre mice occurs when Atm is inactivated. (B) Comparison of brain weight at P7 between Brca2+/+;Nes-cre (Brca2Ctrl), Brca2Nes-cre and Brca2Nes-cre;Atm−/− mice shows that loss of Atm does not restore microcephaly, as brain weight between Brca2Flox/Flox (Brca2Ctrl) and Brca2Nes-cre; Atm−/− is still significantly different (P<0.0001). (C) H&E staining of Brca2Flox/Flox (Brca2Ctrl), Brca2Nes-cre and Brca2Nes-cre;Atm−/− of P21 cerebellum sections shows that development is partially restored by Atm deficiency (magnification × 2); calbindin (D-28K) staining reveals the molecular layer (ML), the Purkinje cell layer (PC) and the inner granule layer (IGL), (magnification × 40). (D) Although proliferation is similar in the EGL of P7 Brca2Ctrl, Brca2Nes-cre and Brca2Nes-cre;Atm−/− cerebella, TUNEL is reduced in the post-mitotic region (parentheses) but not the proliferative layer (asterisk) (magnification × 40); n indicates the number of cerebella analyzed.
Figure 6
Figure 6
Analysis of Brca2Nes-cre neural progenitor cells. (A) Morphology of E14.5 Brca2Ctrl, Brca2Nes-cre and Brca2Nes-cre;p53−/− neurospheres after 7 days in culture. (B) Numbers of E14.5 neurospheres after an initial seeding of 2.5 × 105 cells/ml derived from control or mutant embryos. (C) Numbers of cells present in E14.5 neurospheres derived from control or mutant embryos. (D) BrdU and TUNEL staining of E14.5 neural progenitor cells 3 h after BrdU treatment. (E) Quantitation of BrdU-positive cells in the Brca2Nes-cre neural progenitor cell population compared with Brca2Ctrl, and the respective number of TUNEL-positive cells (F). Asterisks indicate statistically significant differences; n indicates the number of individual cell lines analyzed.
Figure 7
Figure 7
Medulloblastoma occurs in Brca2Nes-cre mice and is associated with p53 deficiency. (A) Kaplan–Meier survival curves of Brca2-deficient mice. Brca2Nes-cre mice were monitored for viability, and >80% died by 32 weeks of age. Brca2Nes-cre mice with associated p53−/− or p53+/− mutations succumbed to medulloblastoma. Total animal numbers are indicated. (B) Examples of typical medulloblastomas in Brca2/p53-deficient mice, represented by a dashed line. Analysis of Brca2Nes-cre;p53−/− medulloblastomas using H&E, Ki-67 or synaptophysin (magnification × 20). (C) SKY analysis of tumors showing translocations and genomic rearrangements on chromosome 11 in Brca2Nes-cre;p53+/− tumors. (D) PCR analysis of p53 showing the loss of p53 WT allele in Brca2Nes-cre;p53+/−; T represents tumor and N represents tail DNA from the same animal.

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