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. 2023 Oct 12:11:1282182.
doi: 10.3389/fcell.2023.1282182. eCollection 2023.

P53 independent pathogenic mechanisms contribute to BubR1 microcephaly

Noelle A Sterling #  1   2 , Bethany K Terry #  1   2 , Julia M McDonnell  1 , Seonhee Kim  1
Affiliations

P53 independent pathogenic mechanisms contribute to BubR1 microcephaly

Noelle A Sterling et al. Front Cell Dev Biol. .

Abstract

The mosaic variegated aneuploidy (MVA)-associated gene Budding Uninhibited by Benzimidazole 1B (BUB1B) encodes BUBR1, a core member of the spindle assembly checkpoint complex that ensures kinetochore-spindle attachment for faithful chromosome segregation. BUB1B mutation in humans and its deletion in mice cause microcephaly. In the absence of BubR1 in mice, massive cell death reduces cortical cells during neurogenesis. However, the molecular and cellular mechanisms triggering cell death are unknown. In this study, we performed three-dimensional imaging analysis of mitotic BubR1-deficient neural progenitors in a murine model to show profound chromosomal segregation defects and structural abnormalities. Chromosomal defects and accompanying DNA damage result in P53 activation and apoptotic cell death in BubR1 mutants. To test whether the P53 cell death pathway is responsible for cortical cell loss, we co-deleted Trp53 in BubR1-deficient cortices. Remarkably, we discovered that residual apoptotic cell death remains in double mutants lacking P53, suggesting P53-independent apoptosis. Furthermore, the minimal rescue of cortical size and cortical neuron numbers in double mutant mice suggests the compelling extent of alternative death mechanisms in the absence of P53. This study demonstrates a potential pathogenic mechanism for microcephaly in MVA patients and uncovers the existence of powerful means of eliminating unfit cells even when the P53 death pathway is disabled.

Keywords: BubR1; DNA damage; cortical development; microcephaly; mitosis analysis; neural progenitor; p53.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Loss of BubR1 causes lagging chromosomes in mitotic neural progenitors. (A) Schematic depicting the method by which dividing neural progenitors at the apical surface of the cortex can be visualized by en face apical explants. (B, C) Representative images and quantification showing an increase in the number of neural progenitors in cytokinesis that display lagging chromosomes in the BubR1 cKO brain. White arrows indicate cytokinetic cells with lagging chromosomes. White boxes indicate cells represented in inset images (B). Scale bar = 50 μm. WT n = 3, BubR1 cKO n = 3. (P = <0.0161). (D–F) Visualization and quantification of mitotic cells displaying DNA damage performed via apical explant staining shows a significant increase in mitotic cells with DNA damage in BubR1 cKO brains. Cells with DNA damage are restricted to prophase where an average of 15% of prophase cells display DNA damage in the BubR1 cKO. Scale bar = 50 μm, 10 μm. WT n = 3, BubR1 cKO n = 3. (PPH3+DNA DAMAGE = <0.0486), (PPH3+DNA DAMAGE PROPHASE = .01100). Data are presented as the mean±SEM and analyzed by a two-tailed Student’s t-test.
FIGURE 2
FIGURE 2
P53 activation occurs in response to BubR1 loss. (A, B) At E14.5, immunohistochemistry, and particle analysis for P21 in a 200 μm width of the cortex demonstrates that BubR1 cKO animals express more P21 protein. This effect can be fully rescued by Trp53 deletion indicating that P53 is activated in response to BubR1 loss. Scale bar = 25 μm. WT n = 6, P53 cKO n = 6, BubR1 cKO n = 3, dcKO n = 3. (ANOVAP21 F = 13.97, df = 3.14, p = 0.0002; Adjusted PWT-p53 CKO = 0.8886, Adjusted PWT−BUBR1 CKO = 0.0001, Adjusted PWT−DCKO = 0.6455, Adjusted PP53 CKO−BUBR1 cKO = 0.0004, Adjusted PP53 CKO−DCKO = 0.9305, Adjusted PBUBR1−DCKO = 0.0036). (C, D) Representative images and quantification of F4/80 labeled activated microglia. Activated microglia are increased in the cortex when BubR1 is lost. This effect is returned to WT levels in the dcKO group, confirming P53 activation when BubR1 is lost. Scale bar = 25 μm. WT n = 5, P53 cKO n = 10, BubR1 cKO n = 3, dcKO n = 7. (ANOVAF4/80 F = 62.25, df = 3.21, P = <0.0001; Adjusted PWT-p53 CKO = 0.4648, Adjusted PWT−BUBR1 CKO = <0.0001, Adjusted PWT−DCKO = 0.9995, Adjusted PP53 CKO−BUBR1 cKO = <0.0001, Adjusted PP53 CKO−DCKO = 0.3107, Adjusted PBUBR1−DCKO = <0.0001).
FIGURE 3
FIGURE 3
At E14.5, microcephaly pathology can be partially improved by Trp53 co-deletion. (A) Representative images showing CC3 labeling of apoptotic cells and MPM2 labelling of mitotic cells in the E14.5 cortex. (B) Quantification of CC3+ apoptotic cells in a 200 μm width of the cortex demonstrates a significant increase in CC3+ cells in BubR1 cKO animals. Cell death still occurs but is significantly reduced in dcKO brains. Scale bar = 25 μm. WT n = 7, P53 cKO n = 10, BubR1 cKO n = 4, dcKO n = 7. (ANOVACC3 F = 107.8, df = 3.24, P = <0.0001; Adjusted PWT-p53 CKO = 0.9686, Adjusted PWT−BUBR1 CKO = <0.0001, Adjusted PWT−DCKO = 0.0002, Adjusted PP53 CKO−BUBR1 cKO = <0.0001, Adjusted PP53 CKO−DCKO = 0.0002, Adjusted PBUBR1−DCKO = <0.0001). (C) Quantification of the distribution of CC3+ dying cells throughout the cortex showing that cell death affects cells in all cortical regions. The proportion of dying cells is not significantly different between any region or between the two animal groups. BubR1 cKO n = 3, dcKO n = 3. (ANOVACC3/Cortical Zone (RowCKO/DCKO: DF=1, F=<.0001, P=>.99)(ColumnRegion: DF=3, F=.6967, P=.6175). (D) Representative images of TUNEL+ cells. Scale bar = 25 μm. (E) Quantification of the proportion of mitotic cells undergoing cell death in a 200 μm width of the cortex at E14.5. The proportion of dying mitotic cells is significantly higher in BubR1 cKO animals compared to WT or P53 cKO. This effect is significantly but not fully reduced in the dcKO group. WT n = 6, P53 cKO n = 6, BubR1 cKO n = 3, dcKO n = 3. (ANOVAMPM2/CC3 F = 29.72, df = 3.14, P = <0.0001; Adjusted PWT-p53 CKO = >0.9999, Adjusted PWT–BUBR1 CKO = <0.0001, Adjusted PWT–DCKO = 0.0120, Adjusted PP53 CKO–BUBR1 cKO = <0.0001, Adjusted PP53 CKO–DCKO = 0.0120, Adjusted PBUBR1–DCKO = 0.0055). (F) Quantification of of TUNEL+ cells in a 200 μm width of the cortex demonstrates a significant increase in TUNEL+ cells in BubR1 cKO animals. Cell death still occurs but is significantly reduced in dcKO brains. WT n = 6, P53 cKO n = 6, BubR1 cKO n = 4, dcKO n = 3. (ANOVATUNEL F = 172.0, df = 3.15, P = <0.0001; Adjusted PWT-p53 CKO = 0.9942, Adjusted PWT–BUBR1 CKO = <0.0001, Adjusted PWT–DCKO = 0.0006, Adjusted PP53 CKO–BUBR1 cKO = <0.0001, Adjusted PP53 CKO–DCKO = 0.0004, Adjusted PBUBR1–DCKO = <0.0001). (G–I) Representative images and quantification of mitotic cells and DNA damage in a 200 μm width of the cortex. Quantification of cells with DNA damage (γH2AX+) at E14.5 in a 200 μm width of the cortex reveals that the number of cells with DNA image is increased in BubR1 cKO but is not improved by Trp53 co-deletion. Scale bar = 25 μm. WT n = 7, P53 cKO n = 10, BubR1 cKO n = 3, dcKO n = 7. (ANOVAγH2AX F = 71.37, df = 3.24, P = <0.0001; Adjusted PWT-p53 CKO = 0.9985, Adjusted PWT−BUBR1 CKO = <0.0001, Adjusted PWT−DCKO = <0.0001, Adjusted PP53 CKO−BUBR1 cKO = <0.0001, Adjusted PP53 CKO−DCKO = <0.0001, Adjusted PBUBR1−DCKO = 0.5170). In BubR1 cKO animals, there is a significant increase in mitotic cells with DNA damage compared to WT that is unchanged by Trp53 deletion. Scale bar = 25 μm. WT n = 6, P53 cKO n = 6, BubR1 cKO n = 3, dcKO n = 3. (ANOVAγH2AX/PH3 F = 28.13, df = 3.14, P = <0.0001; Adjusted PWT-p53 CKO = 0.4383, Adjusted PWT−BUBR1 CKO = <0.0001, Adjusted PWT−DCKO = <0.0001, Adjusted PP53 CKO−BUBR1 cKO = 0.0003, Adjusted PP53 CKO−DCKO = 0.0002, Adjusted PBUBR1−DCKO = 0.9986). (J–K) Quantification of the proportion of γH2AX+ cells with DNA damage that are undergoing apoptosis (CC3+) shows that a significant number of cells with DNA damage are dying in the BubR1 cKO but not the dcKO animals. Scale bar = 25 μm BubR1 cKO n = 4, dcKO n = 7. (p = .0239). White boxes represent inset images. Inset images are closer representations of mitotic cells. Data are presented as the mean±SEM and analyzed by one-way ANOVA followed by a posthoc Tukey test, two-way ANOVA, or a Student’s t-test.
FIGURE 4
FIGURE 4
At E14.5, microcephaly pathology can be partially improved by Trp53 co-deletion. (A) At E14.5 Hematoxylin and Eosin staining demonstrates that ventricular surface length, but not cortical thickness is improved in dcKO cortices compared to BubR1 cKO mice that display a reduced cortex. Scale bar = 100 μm. (B, C) Quantification of ventricular surface length and cortical thickness at E14.5. WT n = 8, P53 cKO n = 8, BubR1 cKO n = 3, dcKO n = 9. (ANOVALENGTH F = 17.29, df = 3.24, P = <0.0001; Adjusted PWT-p53 CKO = 0.2214, Adjusted PWT−BUBR1 CKO = 0.0001, Adjusted PWT−DCKO = 0.3308, Adjusted PP53 CKO−BUBR1 cKO = <0.0001, Adjusted PP53 CKO−DCKO = 0.9892, Adjusted PBUBR1−DCKO = <0.0001). (ANOVATHICKNESS F = 12.38, df = 3.24, P = <0.0001; Adjusted PWT-p53 CKO = 0.3791, Adjusted PWT−BUBR1 CKO = 0.0043, Adjusted PWT−DCKO = 0.0299, Adjusted PP53 CKO−BUBR1 cKO = 0.0002, Adjusted PP53 CKO−DCKO = 0.0005, Adjusted PBUBR1−DCKO = 0.3470). (D, E) Representative images and quantification of PAX6 labeling of neural progenitors at E14.5 in a 200 μm width of the cortex. PAX6+ cell numbers are reduced by BubR1 loss and significantly but not fully rescued in the dcKO. Scale bar = 100 μm. WT n = 6, P53 cKO n = 8, BubR1 cKO n = 3, dcKO n = 5. (ANOVAPAX6 F = 91.02, df = 3.18, P = <0.0001; Adjusted PWT-p53 CKO = 0.2309, Adjusted PWT−BUBR1 CKO = <0.0001, Adjusted PWT−DCKO = 0.0003, Adjusted PP53 CKO−BUBR1 cKO = <0.0001, Adjusted PP53 CKO−DCKO = <0.0001, Adjusted PBUBR1−DCKO = <0.0001). (F, G) Representative images and quantification of SOX9+ neural progenitors in a 200 μm width of cortex at E14.5 shows that cell numbers are significantly reduced by BubR1 loss and are not rescued by subsequent Trp53 co-deletion. Scale bar = 100 μm. WT n = 9, P53 cKO n = 11, BubR1 cKO n = 3, dcKO n = 6. (ANOVASOX9 F = 47.85, df = 3.25, P = <0.0001; Adjusted PWT-p53 CKO = 0.5383, Adjusted PWT−BUBR1 CKO = <0.0001, Adjusted PWT−DCKO = <0.0001, Adjusted PP53 CKO−BUBR1 cKO = <0.0001, Adjusted PP53 CKO−DCKO = <0.0001, Adjusted PBUBR1−DCKO = 0.0003). (H, I) At E14.5, representative images and quantification of the proportion of PAX6+ neural progenitors that are also BrdU+ after 30 min of BrdU incorporation in utero shows that the proportion of S-phase neural progenitors is unchanged by loss of BubR1 or P53. Scale bar = 100 μm. WT n = 6, P53 cKO n = 8, BubR1 cKO n = 3, dcKO n = 5. (ANOVAPAX6/BrdU F = 1.070, df = 3.18, p = 0.3866; Adjusted PWT-p53 CKO = >0.9999, Adjusted PWT−BUBR1 CKO = 0.7737, Adjusted PWT−DCKO = 0.7432, Adjusted PP53 CKO−BUBR1 cKO = 0.7398, Adjusted PP53 CKO−DCKO = 0.7199, Adjusted PBUBR1−DCKO = 0.3191). Data are presented as the mean±SEM and analyzed by one-way ANOVA followed by a posthoc Tukey test.
FIGURE 5
FIGURE 5
Intermediate progenitor and neuron numbers are partially rescued by Trp53 co-deletion at E14.5. (A, B) Representative images and quantification of TBR2 labeling of intermediate progenitors in a 200 μm width of the cortex at E14.5. TBR2+ cell numbers are fully rescued in the dcKO group from their reduced levels in BubR1 cKO cortices. Scale bar = 100 μm. WT n = 7, P53 cKO n = 11, BubR1 cKO n = 3, dcKO n = 3. (ANOVATBR2 F = 10.21, df = 3.20, p = 0.0003; Adjusted PWT-p53 CKO = 0.9522, Adjusted PWT−BUBR1 CKO = 0.0007, Adjusted PWT−DCKO = 0.9957, Adjusted PP53 CKO−BUBR1 cKO = 0.0001, Adjusted PP53 CKO−DCKO = 0.9209, Adjusted PBUBR1−DCKO = 0.0054). (C, D) Representative images and quantification of CTIP2-labeled early-born neurons in a 200 μm width of the cortex at E14.5. The reduction in numbers observed in the BubR1 cKO animals is unaltered in the dcKO. Scale bar = 100 μm. WT n = 9, P53 cKO n = 11, BubR1 cKO n = 3, dcKO n = 6. (ANOVACTIP2 F = 12.17, df = 3.25, P = <0.0001; Adjusted PWT-p53 CKO = 0.5639, Adjusted PWT−BUBR1 CKO = 0.0032, Adjusted PWT−DCKO = 0.0199, Adjusted PP53 CKO−BUBR1 cKO = 0.0003, Adjusted PP53 CKO−DCKO = 0.0008, Adjusted PBUBR1−DCKO = 0.5507). Data are presented as the mean±SEM and analyzed by one-way ANOVA followed by a posthoc Tukey test.
FIGURE 6
FIGURE 6
BubR1 cKO and dcKO show reductions in body and cortex size. (A, B) Representative images and quantification show that BubR1 cKO and dcKO animals are smaller than their WT counterparts at P21. WT n = 3, BubR1 cKO n = 4, dcKO n = 3. (ANOVABODY MASS F = 11.95, df = 2.7, p = 0.0055; Adjusted PWT−BUBR1 CKO = 0.0046, Adjusted PWT−DCKO = 0.0393, Adjusted PBUBR1−DCKO = 0.3436). (C, D) Representative images of whole brains and quantification of cortical area at P21 shows an overall cortical reduction in BubR1 cKO animals that is unchanged in dcKO animals. WT n = 4, P53 cKO n = 4, BubR1 cKO n = 3, dcKO n = 3. (ANOVACORTICAL AREA F = 251.8, df = 3.10, P = <0.0001; Adjusted PWT-p53 CKO = 0.6042, Adjusted PWT−BUBR1 CKO = <0.0001, Adjusted PWT−DCKO = <0.0001, Adjusted PP53 CKO−BUBR1 cKO = <0.0001, Adjusted PP53 CKO−DCKO = <0.0001, Adjusted PBUBR1−DCKO = 0.3496). Data are presented as the mean±SEM and analyzed by one-way ANOVA followed by a posthoc Tukey test.
FIGURE 7
FIGURE 7
Concurrent Trp53 deletion only partially rescues microcephaly pathology in BubR1 cKO animals at P21. (A) Hematoxylin and Eosin staining shows that BubR1 cKO causes microcephaly at P21 which is minimally improved in dcKO animals. Scale bar = 500 μm. Red dotted lines denote where the ventricular surface area was measured. Black lines indicate where cortical thickness was measured. (B, C) Quantification of ventricular surface length and cortical thickness at P21 indicates a mild rescue of ventricular length but not cortical thickness in the dcKO compared to BubR1 cKO. WT n = 5, P53 cKO n = 5, BubR1 cKO n = 4, dcKO n = 5. (ANOVA LENGTH F = 47.50, df = 3.15, P = <0.0001; Adjusted PWT-p53 CKO = 0.9870, Adjusted PWT−BUBR1 CKO = <0.0001, Adjusted PWT−DCKO = <0.0001, Adjusted PP53 CKO−BUBR1 cKO = <0.0001, Adjusted PP53 CKO−DCKO = <0.0001, Adjusted PBUBR1−DCKO = 0.0246). (ANOVA THICKNESS F = 21.53, df = 3.15, P = <0.0001; Adjusted PWT-p53 CKO = 0.6554, Adjusted PWT−BUBR1 CKO = 0.0022, Adjusted PWT−DCKO = 0.0003, Adjusted PP53 CKO−BUBR1 cKO = 0.0003, Adjusted PP53 CKO−DCKO = <0.0001, Adjusted PBUBR1 DCKO = 0.8940).) (D–F) Immunohistochemistry for FOXP2, CTIP2, and CUX1 demonstrates that BubR1 cKO causes a reduction in both early-born and late-born populations of neurons, while concurrent Trp53 deletion minimally improves cell loss. Scale bar = 100 μm. (G) Quantification of cortical neuron numbers at P21 in a 400 μm width of the cortex. (FOXP2: WT n = 4, P53 cKO n = 5, BubR1 cKO n = 4, dcKO n = 7. ANOVAFOXP2 F = 5.162, df = 3.16, p = 0.0875; Adjusted PWT-p53 CKO = >0.9999, Adjusted PWT−BUBR1 CKO = 0.0222, Adjusted PWT−DCKO = 0.9455, Adjusted PP53 CKO−BUBR1 cKO = 0.0154, Adjusted PP53 CKO−DCKO = 0.9330, Adjusted PBUBR1−DCKO = 0.0287). (CTIP2: WT n = 5, P53 cKO n = 7, BubR1 cKO n = 5, dcKO n = 5. ANOVACTIP2 F = 22.34, df = 3.18, P = <0.0001; Adjusted PWT-p53 CKO = 0.8262, Adjusted PWT−BUBR1 CKO = <0.0001, Adjusted PWT−DCKO = 0.0569, Adjusted PP53 CKO−BUBR1 cKO = <0.0001, Adjusted PP53 CKO−DCKO = 0.0006, Adjusted PBUBR1−DCKO = 0.1781). (CUX1: WT n = 5, P53 cKO n = 7, BubR1 cKO n = 5, dcKO n = 5. ANOVACUX1 F = 478.3, df = 3.18, P = <0.0001; Adjusted PWT-p53 CKO = 0.1933, Adjusted PWT−BUBR1 CKO = <0.0001, PWT−DCKO = <0.0001, Adjusted PP53 CKO−BUBR1 cKO = <0.0001, Adjusted PP53 CKO−DCKO = <0.0001, Adjusted PBUBR1−DCKO = 0.0111). Data is presented as the mean ±SEM and analyzed by one-way ANOVA followed by a posthoc Tukey test.
FIGURE 8
FIGURE 8
Trp53 co-deletion does not significantly rescue hippocampal reduction in BubR1 cKO at P21. (A) Hematoxylin and Eosin staining and shows that the size of the hippocampus is reduced in both BubR1 cKO and dcKO animals. Scale bar = 100 μm. (B) Quantification of hippocampal area shows that hippocampal size is significantly reduced by BubR1 loss and not restored by subsequent Trp53 co-deletion. WT n = 5, P53 cKO n = 5, BubR1 cKO n = 4, dcKO n = 5. (ANOVAAREA F = 107.9, df = 3.15, P = <0.0001; Adjusted PWT-p53 CKO = 0.1147, Adjusted PWT−BUBR1 CKO =<0.0001, Adjusted PWT−DCKO = <0.0001, Adjusted PP53 CKO−BUBR1 cKO = <0.0001, Adjusted PP53 CKO−DCKO = <0.0001, Adjusted PBUBR1−DCKO = 0.8009). (C, D) Representative images and quantification of CTIP2+ neurons in the hippocampal CA1/CA2 region at P21 show a significant reduction in cell numbers caused by BubR1 loss that is unimproved in the dcKO brain. Scale bar = 100 μm. WT n = 4, P53 cKO n = 6, BubR1 cKO n = 5, dcKO n = 5. (ANOVACTIP2 F = 26.76, df = 3.16, P = <0.0001; Adjusted PWT-p53 CKO = 0.4990, Adjusted PWT−BUBR1 CKO = 0.0001, Adjusted PWT−DCKO = 0.0049, Adjusted PP53 CKO−BUBR1 cKO = <0.0001, Adjusted PP53 CKO−DCKO = 0.0001, Adjusted PBUBR1−DCKO = 0.2595). Data are presented as the mean±SEM and analyzed by one-way ANOVA followed by a posthoc Tukey test.
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