Therefore, proper DNA repair pathways are essential to counteract various endogenous DNA damages that continually compromise genomic integrity. Atad5 is important for maintaining genomic integrity by regulating PCNA unloading from chromatin using its ATPase activity during replication. Therefore, to understand the role of the Atad5 gene in brain development, we generated Atad5 brain-specific knockout mice using the Nestin-Cre recombinase system.
Atad5 brain-specific knockout mice were viable, but body weight and brain size were significant in adult mice. Consistently, p53, which is an important regulator of DNA damage response, was upregulated in the Atad5 brain-specific knockout mouse. Taken together, our data suggest that Atad5, which has a PCNA unloading function, has a role in brain development.
List of abbreviation
Introduction
Atad5 is important for maintaining genomic integrity in eukaryotic organisms (Bell et al., 2011; Fox, Lee, & Myung, 2011). Atad5 has a major role in PCNA (proliferating cell nuclear antigen) unloading and PCNA deubiquitination (Fox et al., 2011). Atad5-depleted cells showed sensitivity to DNA damaging agents and genomic instability (Bell et al., 2011; Fox et al., 2011).
In particular, Atad5-depleted cells have shown that the failure of proper termination of repair DNA synthesis, and frequent notch exposure by H2O2 treatment generating ROS-induced SSBR (Park et al., 2021). Consistent with our hypothesis, 90% of Atad5 haploinsufficient mice developed multiple tumors in many different tissues, and their tumors showed high levels of genomic instability ( Bell et al., 2011 ). Atad5 homozygous mutant mice were embryonic lethal, implying that Atad5 plays a critical role in embryonic development ( Bell et al., 2011 ).
Because Atad5 homozygous mutant mice died before neurogenesis, we could not investigate the role of Atad5 in brain development. In previous studies, Atad5 is highly expressed in the brain during zebrafish embryogenesis ( Shin et al., 2020 ). Furthermore, DNA damage was increased and p53-dependent apoptosis occurred through ATM-Chk2 signaling in the developing brain of Atad5 mutant zerafish ( Shin et al., 2020 ).
The brain phenotype of the Atad5 zebrafish mutant supports our hypothesis of the involvement of Atad5 in mouse brain. Therefore, the role of Atad5 in mouse brain development and how Atad5 affects the adult brain through failure of neurogenesis are still unknown. To study the role of Atsd5 in vivo, we generated Atad5 brain-specific knockout mice by Nestin-Cre, which expresses the Cre recombinase in the central nervous system.
Atad5-specific depletion in the brains of mice showed a reduction in body weight and brain size. The number of cells in the hippocampus and cerebellum was significantly reduced in the Atad5 brain-specific knockout mice. Consistently, the p53 level was induced in the brains of the Atad5 brain-specific kncokout mice.
Materials and Methods
Adult mouse ovary and testis were fixed in 10% neutral buffered formalin (NBF) and embedded in paraffin blocks. Adult brains were fixed in 4% PBS-buffered paraformaldehyde by intracardiac perfusion and immersion fixed in 4% PBS-buffered paraformaldehyde for 15 h, fixed tissues were washed with PBS for three times and cryoprotected in PBS-buffered 30% sucrose solution. Adult brains were sectioned in sagittal fashion and stained with hematoxylin and eosin (H&E).
Whole brains were homogenized in Trizol (Ambion) and extracted according to the manufacturer's instructions. cDNA was synthesized with SuperScriptTM Ⅳ (Invitrogen). To measure mouse skull size in vivo, mice were anesthetized with 1~2% isoflurane during μCT scanning. For the cranial to caudal direction, the lengths from the caudal end of the skull to lambda, from lambda to bregma, or from bregma to the beginning of the frontal bone were defined as CC1, CC2, and CC3, respectively.
For the left-to-right direction, the left-to-right lengths in the lambda or bregma cross-section were defined as LR1 or LR2. For the ventral to dorsal direction, the vertical lengths from basisphenoid bone to lambda or bregma were defined as VD1 or VD2, respectively. ROIs were manually drawn on MRI or μCT images and brain volume is calculated based on Equation 1.
Where ROI is a manually drawn 3D binary matrix, Vx, Vy, and Vz are the length of the voxel in the x, y, and z directions, respectively.
Results
In addition, we confirmed that Atad5 mRNA level was decreased to 0.2-fold only in the brains of NesCre;Atad5flox/flox mice by RT-qPCR ( Fig. 1E ). Taken together, Atad5 was only depleted in the brains of NesCre;Atad5flox/flox mice by the Nestin-Cre system. Weights of NesCre;Atad5flox/flox mice were monitored in juvenile (1–2 month), young adult (3–6 month), and adult (7–13 month) groups.
The brain ratio of NesCre;Atad5flox/flox mice was smaller than that of NesCre;Atad5wt. Furthermore, the brain weights of NesCre;Atad5flox/flox mice were significantly lighter than those of NesCre;Atad5wt after deletion of Atad5 (wt; 0.48 g mt: 0.38 g) (Fig. 2B). The increase in DNA damage in NesCre;Atad5flox/flox could lead to a reduction in brain size.
Therefore, we checked the p53 mRNA level and it was mildly upregulated in NesCre;Atad5flox/flox mice (Fig 2C). All age groups of NesCre;Atad5flox/flox mice were lighter than NesCre;Atad5wt. Brain length of NesCre;Atad5flox/flox/total length (%) mice was smaller and brain weight was lighter compared to NesCre;Atad5wt mice.
P53 expression is mildly induced in 1-year-old whole-brain NesCre;Atad5flox/flox mice. Furthermore, we observed that the ovary of NesCre;Atad5flox/flox mice has more pyknotic nuclei, indicating damaged cells compared to NesCre;Atad5wt (Fig 3E). NesCre;Atad5flox/flox mice revealed that the total brain volume was much smaller than in NesCre;Atad5wt mice at 8 weeks and 1 year of age (Fig. 4A).
All of the brain sections of each segmented NesCre;Atad5flox/flox mouse were smaller than NesCre;Atad5wt (Fig. 4B). The total brain volume of NesCre;Atad5flox/flox mice was smaller than NesCre;ATAD5wt mice ****P<0.0001 (t-test). In the hippocampus, the cell numbers of cornu ammonis1 (CA1) and cornu ammonis3 (CA3) were reduced, and the thickness of the dentate gyrus (DG) was reduced in the NesCre;Atad5flox/flox mice brains *P<0.05, **P< 0.01 (t-test).
However, NesCre;Atad5flox/flox embryos did not show strong morphological differences compared to NesCre;Atad5wt (Fig 6. A,B). This differential gene expression pattern of NesCre;Atad5flox/flox suggested that depletion of Atad5 affects neuronal differentiation in embryonic brain.
Discussion
One of the roles of p53 is to maintain a balance between self-renewal and differentiation for proper development and maintenance of tissue homeostasis (Armesilla-Diaz et al., 2009). During neurogenesis, genomic stability is important for proliferation and self-renewal (Pearson et al., 2020; Sanchez-Ortiz et al., 2014). In previous studies, p53 depletion in olfactory bulb-derived neurospheres showed increased self-renewal and proliferation ( Sanchez-Ortiz et al., 2014 ).
Our data showed that Atad5 mutant brain at E16.5 showed increased level of p53 and pax6 and decreased level of Tbr1, suggesting that self-renewal may not be defective, but the differentiation process is delayed and/or defective in the absence of Atad5. Mutation of ATM usually causes microcephaly, and it is highly related to cerebellar defects (Ding et al., 2021; J. Kim et al., 2020). Atad5 knockout zebrafish embryo showed that ATM/Chk2 signaling induces p53-dependent apoptosis in the brain and this causes brain size reduction (Shin et al., 2020). Our data showed that microcephaly in adult brain and p53 was induced by ATAD5 depletion in the brain.
On the other hand, during brain development, large amounts of ROS are produced and cause single-strand breaks (SSBs). In previous studies, depletion of Xrcc1, which is critical for SSB reaper in the mouse brain, failed to proliferate and mature in a p53-dependent manner in cerebellar interneurons (Lee et al., 2009). In addition, ATAD5-depleted human cells showed defects in SSB repair resulting from oxidative DNA damage.
Since Atad5 mutant brains show defects in the cerebellum, it will be interesting to test whether SSB accumulates and affects proliferation.
