Thegenome is the genetic material for all living organisms. It is the entire set of hereditary information for building, running, and maintaining an organism and transmitting its informationtonext generation. Transmission of genetic information is constantly in a selective balance between the maintenance of genetic stability and elimination of mutational change. The genome is made of chemical DNA (Deoxyribonucleic acid). The DNA molecule is being continuously exposed to various types of damage caused by exogenous source (e.g.
ionizing radiation, ultraviolet light from sunlight, chemicals, and pollutants) and endogenous sources (e.g. stalled forks during DNA replication and reactive oxygen species during respiration) during our lifetime. From the structural point of DNA damage lesions, it can be divided into two types of DNA damage such asDNA single strand break and DNA double strand breaks under large perspective (De Bont and van Larebeke, 2004; Lopez-Contreras and Fernandez-Capetillo, 2012). The quantity of lesions that every cell faces is up to 105 spontaneous or induced DNA lesion per day (Ames et al., 1993; Nakamura et al., 1998;
Vilenchik and Knudson, 2003; De Bont and van Larebeke, 2004). It estimates that each individual could deal withmore than 105spontaneous or induced DNA lesions per day. To counteract DNA damage, eukaryotic cells have developed a complex signaling network of repair processes known as DNA damage response to recruit and activate the right factors in the right place at the right time (Ciccia and Elledge, 2010).
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DNA repair is extremely important in the nervous system because DNA damage occurs spontaneously during brain development and in the adult brain, (Lu et al., 2004; Shull et al., 2009; Suberbielle et al., 2013). In the nervous system unrepaired DNA lesions and mutations can have an enormous effect on the formation of a functional nervous system (McKinnon, 2013). Defects in cellular DNA damage signaling and repair processes can lead to many human hereditary syndromes(Table 1). Representatively, Ataxia oculomotor apraxia (AOA1) and Spinocerebellar ataxia with axonal neuropathy (SCAN1) are related to disfunctional DNA single strand break repair proteins. AOA1 and SCAN1 result from mutations in the Aprataxin (APTX) and Tyrosyl-DNA phosphodiesterase 1 (TDP1) genes respectively. APTX acts for repairing 5’-end resulting from abortive DNA ligation reactions. TDP1 is an enzyme required for the 3’-end tyrosyl DNA procession after abortive Topoisomerase 1 (Top1) mediated torsional stress induced DNA single strand break. Theconsequence of defects in APTX orTDP1 ischaracterized by cerebellarataxia and sensorimotor neuropathy (McKinnon and Caldecott, 2007; Rass et al., 2007). An interesting feature is that the pathology in these disorders is almost exclusively restricted to the nervous system implying the importance of maintaining genomic stability during brain development.
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Table 1. Human neurological diseases linked to mutation in DNA repair gene and its mouse model
The reports of mouse model are listed below.
1)(El-Khamisy et al., 2009), 2) (Katyal et al., 2007), 3) (Barlow et al., 1996), 4) (Buis et al., 2008), 5) (de Klein et al., 2000), 6) (Murga et al., 2009), 7) (Zhu et al., 2001),
8) (Gomez-Herreros et al., 2014)
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Ataxia telangiectasia (A-T) is another prime example of the relationship between DNA damage responsedefect and neurodegenerative disorder. A-T is caused by mutation in the ATM gene (Ataxia Telangiectasia mutated). ATM is a serine/threonine (Ser/The) protein kinase and belongs to the phosphoinositide 3-kinase (PI3K) family, which is also includes ATR (Ataxia telangiectasia and Rad3-related protein) and DNA-PKcs (catalytic subunit of DNA-dependent protein kinase) (Savitsky et al., 1995). These two proteins are also involved in DNA damage responses. ATM is rapidly recruited to DNA double strand break (DSB) sites and phosphorylates many downstream substrates to activate the cellular DSB responses including DNA damage repair, cell cycle checkpoint and apoptosis (Shiloh and Ziv, 2013).
The well-known tumor suppressor; p53 was the first substrate of ATM identified in vitro and in vivo(Banin et al., 1998; Canman et al., 1998; Khanna et al., 1998). In A-T cells (ATM deficient human cells), the stabilization and activation of p53 was defected, and these cells were characterized by defective G1-S checkpoint, in which p53 has a central role (Kastan et al., 1992; Khanna and Lavin, 1993).
A-T is an autosomal recessive disorder that is estimated to affected 1 in 40,000-100,000 people (Crawford, 1998). It has been reported that the 432 different mutations in the ATM gene without any hotspots(Lavin et al., 2007). This is an early childhood onset disease at aroundage 12 months-2 years, when ataxia of gait and posture is an important sign of the clinical features as the child begins to walk (Boder and Sedgwick, 1958). The clinical features of A-T patients are sterility, chromosomal instability, immunodeficiency and neurologic impairments including cerebellar ataxia and abnormal eye movement (oculomotor apraxia). Affected persons also manifest both sensitivity to ionizing radiation
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and cerebellar atrophy (Taylor et al., 1975; Cabana et al., 1998; Crawford et al., 2000). In many clinical reports, A-T patients showed dysmyelinated white matter in the brain called leukodystrophy (Chung et al., 1994; Sardanelli et al., 1995; Opeskin et al., 1998; Habek et al., 2008). Up to 30% of A-T patients develop lymphoid tumors since ATM plays a critical role in the differentiation of T and B cells (Lumsden et al., 2004). At present there is no cure and no way to slow the neuropathological progression of the A-T patients. It is possible to alleviate some of the symptoms associated with immunodeficiency (Lavin et al., 2007).
A slow progress has been made in the research of neuropathologicalphenotypes of human A-T patients. A great deal of knowledge to understand neurological defects in human diseases comes from analyzing proper animal models that mimic neurological phenotypes in human. However, unfortunately most of animal models for A-T did not show any sign of neuropathology such as ataxia and neurodegeneration. One of possibilities is that the kind of endogenous DNA damage to induce ATM signaling is different from what has been reported.
To investigate the ATM function for maintaining genomic stability during brain development, we generated anXrcc1 and Atmdouble inactivatedmouse model using a tissue specific gene deletion system. We hypothesized that chronic DNA damage by Xrcc1 inactivation to block both base excision repair (BER) and DNA single strand break repair (SSBR) triggers Atm dependent signaling for homeostasis during brain development. Xrcc1 interacts with various DNA repair factors, including PARP1, APTX, DNA polymerase β, DNA ligase 3 and indirectly TDP1(McKinnon and Caldecott, 2007). Inactivation of Xrcc1 couldinducechronic genotoxic stress due to unrepaired endogenous DNA damage during brain development (Lee et al., 2009). Bysimultaneous inactivation of Xrcc1 and Atm in the
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mouse, we examined the function of Atmto maintain genomic stability during brain development. We found that Atmis required for proper formation of the cerebellar structure and full differentiation of oligodendrocytes. We also found that in cerebellar interneuron progenitor cells, p53, one of the ATM substrates, could be regulatedby another protein kinase rather than ATM upon DNA damage induced by XRCC1 deficiency. The more important point we want to make clear is that in contrast with existing A-T mouse models, our Xrcc1 and Atmdouble knockout mouse showed many similar neurological phenotypesto those observed inhuman A-T patients. Our new A-T mouse model is quite valuable to reveal the molecular mechanisms and etiology of A-T neural phenotypes, and to establish brain specific cellular function of ATM and its signaling pathway.
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II.MATERIALS AND METHODS
A. Generation of Atmand Xrcc1 knockout animal model
Atm and Xrcc1 mouse lines have been previously reported (Herzog et al., 1998; Lee et al., 2009). To inactivate simultaneously the Atmand Xrcc1 genes in the nervous system, we bred Xrcc1loxP/loxP; ATM+/- with Nes-cre mice. Xrcc1Nes-cre; Atm-/- mice were obtained by crossing male Xrcc1loxP/loxP; Atm+/-;orXrcc1loxP/+; Atm+/- mice and femaleXrcc1loxP/loxP; Atm+/- ;Nestin-cre orXrcc1loxP/+; Atm+/-; Nestin-cremice. We maintained Nes-creexpression only in females to avoid ectopic expression of Cre recombinase.Littermates were used as controls including Xrcc1+/+; Atm+/+(wildtype: WT), Xrcc1loxP/+; Atm+/+, Xrcc1loxP/loxP; Atm+/+, Xrcc1+/+; Atm+/-, and Xrcc1loxP/+; Atm+/-.Atmheterozygotes (Atm+/-)and Xrcc1loxP+; Nes-cre animals did not show any difference from WT. The presence of a virginal plug was considered embryonic day 0.5 (E0.5) and the day of birth as postnatal day 0 (P0).
The genotypes of the mice were determined by PCR (polymerase chain reaction). Primer sequences for the Xrcc1, Cre, andAtmgenes are listed in below (Table 2).
l PCR for the Xrcc1 gene
WT (Xrcc1+/+) and Floxed (Xrcc1loxP/loxP) alleles
A combination of primers is PC1 and PC2:WT (245bp) and the Xrcc1loxP/loxPgene (279bp) PCR products were amplified for 35cycles of 94℃ for 30 sec, 58℃ for 45 sec and 72℃
for 45 sec.
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l PCR for the Cre recombinase gene Cre recombinaseallele
A combination of primers isCre3 and Cre4
PCRproduct were amplified for 36 cycles of 94℃ for 30 sec, 60℃ for 45 sec and 72℃
for 45 sec.
l PCR for the Atm gene WT allele of the Atm gene
A combination of primers Atm WT1 and WT2
PCRproduct were amplified for 36 cycles of 94℃ for 1 min, 60℃ for 1 min and 72℃
for 1 min.
Mutant allele of the Atm gene
A combination of primers Atm Atm4380 and Neo5
PCRproduct were amplified for 36 cycles of 94℃ for 1 min, 55℃ for 1 min and 72℃
for 1 min.
９ Gene Primer Primer sequence
XRCC1 PC1 5’-TAT GCT TGC TGT ACA GGG ATT GGG-3’
PC2 5’-TGG ACC ATG AAA AAG CTG TGT GC-3’
Cre Cre3 5’-CTG CCA CGA CCA AT GAC AGC-3’
Cre4 5’-ACC TGC GGT GCT AAC CAG CG-3’
Atm WT WT1 5’-GCC TGT TAT CTT CTA TGT GCA CCG TCT TCG C-3’
WT2 5’-GGT GCG GTG TGG ATG GGA CTG GAG G-3’
Atm Mutant Atm4380 5’-GTG ATG GAC CTG AGA CAA GAT GCT GTC-3’
Neo 5 5’-GGG AAG ACA ATA GCA GGC ATG C -3’
Table 2. List of primer used for PCR based Genotyping
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B. Isolation and differentiation of primary mouse neural stem cell.
Primary neural stem cells (NSC) were purified from embryonic brains at embryonic day 14 (E14.5).The embryonic forebrain was dissected after removingthe scalp, skull and meninges.
The isolated forebrain was triturated mechanically into a single cell-suspension using 1ml micropipette set at approximately 950μl, 200μl micropipette set at approximately 190μl and polished Pasteur pipettes gently by piptetting up and down 20 times each. Single NSCs were cultured in neural stem cell basal medium (STEM CELL technology) supplemented with proliferation supplements (STEM CELL technology) and 20ng/mlEpidermal growth factor (EGF, STEM CELL technology) in T-25 tissue culture flasks (SPL) until neurospheres were established. Neurospheres were split every 6~7 days. For in vitroNSC differentiation assay, E14.5 neural stem cells were cultured at 1× L-ornithine (Sigma) coated plate and in neural stem cell basal medium (STEM CELL technology) supplemented with differentiation supplements (STEM CELL technology). Differentiation medium was partially replaced every 2~3 days. The status of differentiation was checked on DIV (days in vitro) 14 or DIV 21. Both proliferation and differentiation culture were established at 37 ℃ in a humidified, CO2-regulated (5%) incubator.
C. Determination of cell number in proliferation
Neural stem cells were plated at 5x104cells/well in neural stem cell basal medium supplemented with proliferation supplement and 20ng/mlEpidermal growth factor (EGF,
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STEM CELL technology). Neurospheres were collected at Day 7 after plating and then dissociated to the single cell level, and total single neural stem cell numbers were counted using a hemocytometer.
D. Antibodies for histology and western blot
For immunohistochemistry and immunocytochemistry, we used antibodies to calbindin D28K (mouse, 1:2000, Sigma), GABA receptor α6 (rabbit, 1:500, Chemicon), GFAP (mouse 1:1000, sSigma), Pax2 (rabbit, 1:500, Zymed), gH2AX (rabbit, 1:500, Abcam), 53BP1 (rabbit, 1:500, Bethyl Laboratory), PCNA (mouse, 1:500, Santa Cruz), Olig2 (rabbit, 1:1000, Millipore), PDGFRα (rabbit, 1: 1000, Santa Cruz), MBP (rabbit, 1:500, Abcam). The antibodies listed above were used with a citrate buffer based antigen retrieval treatment.
This antibody was used without antigen retrieval: Olig1 (rabbit, 1:1000, LS Bio).
For western blot, we used antibodies to NeuN clone A60 (mouse 1:1000, Millipore), Actin (mouse, 1:5000, Abcam), CNPase (mouse, 1:1000, Sigma), Olig1 (rabbit, 1:1000, LS Bio), Olig2 (rabbit, 1:1000, Millipore).
E. Immunohistochemistry
Embryos and brain tissues were removed at indicated ages after cardiac perfusion with 4%
(wt/vol) buffered paraformaldehyde, cryoprotected in buffered 25% sucrose (wt/vol) solution and sectioned at 10 μm using an MEV cryostat (SLEE).
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Immunohistochemical analysis of tissue and cells, were perform using the antibodies listed above. Sections were incubated with antibodies overnight after quenching endogenous peroxidase using 0.6% (vol/vol) H2O2 in methanol. After washing slides with Phosphate-buffered saline (PBS) two times, the tissues were treated with biotinylated secondary antibody (Jackson ImmunoResearch) and avidin-biotin complex (Vector Labs).
Immunoreactivity was amplified with VIP substrate kit (Vector labs). Sections were counterstained with 0.5% methyl green, dehydrated and mounted in DPX (Sigma). For fluorescence immunoreactions of sections, FITC- or Cy3 conjugated Secondary antibodies (Jackson ImmunoResearch) were used and counterstained with DAPI (Vector Labs). All stained slides were examined and imaged using an OPTIKA B-600TiFL equipped with a Leica DFC310 FX digital camera.
F. TUNEL assay
Embryos and brain tissues were labeled with TdT enzyme and Anti-digoxigenin conjugate antibody to detection of cell death using ApopTag®Fluorescine In situ Apoptosis Detection Kit S7110 (Millipore) following manufacturer’s instructions.
G. Protein extraction (nuclear/cytosol) and western blot analysis
Protein extracts (from cells or tissues) were prepared by using lysis buffer (500mM
Tris-１３
HCl, 150mM NaCl, 50mM NaF, 1% Tween-20 (vol/vol), 0.2% NP-40, 1mM DTT,phosphatase inhibitor cocktail 2 (Sigma), Protease inhibitor tablet (Sigma)).
AlsoNucleus and Cytosol protein were extracted from P14 mouse brains using Nuclear/Cytosol Fractionation Kit (Biovision). All protein samples were quantified by Bradford assay (Bio-Rad).Proteins (30μg per lane or 50μg per lane) were separated through a Bis-Tris 10% (wt/vol) polyacrylamide gel and transferred in to nitrocellulose membrane (GE Healthcare).
Primary antibodies were listed above. Secondary antibodies were used Goat- anti mouse, anti- rabbit HRP-conjugated antibody (Bio-Rad) and detected using ECL (Bio-Rad). Actin antibody (1:2500, Santa Cruz Biotech) served as a protein-loading control. For the nuclear extracts, H3 antibody was used for fractionation efficiency and loading control.
H. RNA extraction and quantitative real time PCR
Total RNA was extracted from the brain tissue or Neural stem cells using an TRIzol reagent (Invitrogen). To generate cDNA, 4 μg of total RNA was used in 20 μl of revers transcription reaction, using Super-ScriptⅢ RT-PCR Kit (Invitrogen). Quantitative real time
PCR was performed using Rotor-Gene SYBR Green RT PCR Kit (Qiagen). Data were normalized to the internal control b-actin and analyzed DDCt method. Primer list used in quantitative PCR for mouse are given in below table (table 3).
１４ Gene Primer Primer sequence
β-actin Forward 5’-CAG CAA GCA GGA CTA CGA TGA G-3’
Reverse 5’-CAG TAA CAG TCC GCC TAG AAG CA-3’
PDGFRα 1 Forward 5’-CAA ACC CTG AGA CCA CAA TG-3’
Reverse 5’-TCC CCC AAC AGT AAC CCA AG-3’
MBP 2 Forward 5’-CTA TAA ATC GGC TCA CAA GG-3’
Reverse 5’-AGG CGG TTA TAT TAA GAA GC-3’
PLP 3 Forward 5’-AGC AAA GTC AGC CGC AAA AC-3’
Reverse 5’-CCA GGG AAG CAA AGG GGG-3’
Table 2. List of primers for Quantitative real time PCR The reports of primers are listed below
1)Ning Zhang, J.Biol. Che, 2009 2) Hai Jie Yang, J Mol Neurosci, 2014 3) Marcel Tawk, J. Neurosci, 2011
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III.RESULTS
1.Double inactivation of Xrcc1 and Atm in the central nervous system
Deletion of theXrcc1 gene increased the number of endogenous DNA Single strand breaks(Khoronenkova and Dianov, 2015). To determine the role of ATM upon DNA damage during brain development, we generatedXrcc1 and Atm double knockout mice.Xrcc1 germline knockout mice showed early embryonic lethality before brain development (Tebbs et al., 1999). To resolve this, we used a Nestin-Cre system to generate Xrcc1conditional knockout mice which induce tissue specific DNA damage, particularlyin the central nervous system during development.This Xrcc1conditional knockout mouse modelhasbeen previously described (Lee et al., 2009). Xrcc1conditionally targeted mice (hereafter referred to as Xrcc1nes-cre) were crossbred with Atm germline knockout mice (hereafter referred to as Atm-/-) (Herzog et al., 1998)(Figure 1). Generated Xrcc1 and Atm double knockout mice (hereafter referred to as Xrcc1nes-cre; Atm-/-) died beforepostnatal week 3. The genotype of mouse in these experimentswas confirmed at the DNA level (Figure 2A). Xrcc1nes-cre; Atm -/-mice had smaller brain than those of littermates of WT and Atm-/- especially the cerebellum (Figure 2B). Gross behavior observation indicated that unlike WT and Atm-/- mice, Xrcc1
nes-creanimals showed mild ataxia with episodic spasms as previously reported(Lee et al., 2009).
StrikinglyXrcc1nes-cre; Atm-/- animals showed severe ataxia and exacerbated neurological phenotypeswhich were not observed either in Xrcc1nes-creor Atm-/-animals (Figure. 2C).
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Figure 1. Strategy of generatingXrcc1 and Atm knockout mouse model.
(A) The conditional targeting construct for the Xrcc1 gene was engineered with loxP sites flanking exons 4-10. The Atm germline targeting construct was engineered with Neo gene to interrupt exon 57 and replace a part of exon 58 of the Atm gene. The targeted region is the phosphoinositol 3-kinase domain (PI3K domain) which is catalytic activity domain of Atm.
(B) To inactivate the Xrcc1 and Atmgene in the nervous system, first we gained the Xrcc1loxP/+;Atm+/- mice. And then Xrcc1loxP/loxP;Atm-/-;Nestin-Cre+mice were obtained bybreeding male Xrcc1loxP/+;Atm+/-with femaleXrcc1loxP/+;Atm+/-;Nestin-Cre+mice.We maintained Nestin-Crein female mice to excludeectopic Cre recombinase activity.
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Figure 2. Generation of an Xrcc1 and Atm double knockout mouse.
(A) PCR based genotyping showed that Xrcc1 and Atm double knockout mouse has a LoxP floxed Xrcc1, Atm neo-cassette (Atm mutant) and Cre recombinase alleles. (B) Small brains were found in postnatal day 15 (P15)Xrcc1Nes-Creanimals and much smaller brains in Xrcc1
Nes-Cre; Atm-/- mice compared with those of WT and Atm-/- mice. (C) Comparative view of WT and Xrcc1Nes-Cre; Atm-/- mice. Xrcc1Nes-Cre; Atm-/- mice (red arrows) showed exacerbated neurological behavior phenotypes such as progressive severe ataxia.
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2. Loss of Xrcc1 and Atm results in DNA repair deficiency in the brain
Xrcc1 and ATM are critical for DNA single strand break repair and double strand break repair respectively. To estimate the role of Xrcc1 and Atmin repairing DNA damage in the nervous system, we analyzed the brains of Xrcc1nes-cre; Atm-/-mice for the accumulation of DNA strand breaks. We used gH2AXas a marker for DNA damage that could be detected as foci formation in the nucleus. gH2AXis the phosphorylatedform of histone H2AXthatis accumulated at the sites of DNA strand breaks to act as a sensor for DNA damage and to initiate DNA damage repair responses(Rogakou et al., 1998).Although gH2AX typically involves in DNA double strand breaks, DNA single strand breaks by Xrcc1 deficiency can be converted to DNA double strand break that arise during replication or random damage accumulation leading to adjacent breaks.
During early neural development,there was widespread accumulation of DNA damage throughout theXrcc1nes-cre; Atm-/-mouse brain more than that of the Xrcc1nes-cre brain.gH2AXfoci formation occurred in proliferating cells in the various areas of both Xrcc1nes-creand Xrcc1nes-cre; Atm-/- mice brain.WhenXrcc1nes-creand Xrcc1nes-cre; Atm-/- animals were compared,the Xrcc1nes-cre; Atm-/-brain showedmore accumulation of gH2AX signal than the Xrcc1nes-crebrain, suggesting that Xrcc1nes-cre; Atm-/- animals faced increased challenge to DNA damage and by inactivating Atm, the load of DNA damage was affected (Figure 3A,B).
Moreover, these DNA strand breakswas not repaired until proliferating cells becoming post-mitotic differentiated cells.WT andAtm-/-mice brain did not show any noticeable gH2AX positive signal besides small gH2AX foci that occur during normal proliferation (data not
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shown).This sign of DNA damage accumulation was also confirmed with 53BP1 foci;
another DNA damage marker (Figure 3A). 53BP1 is one of many early responding proteins to DNA damage which is phosphorylated upon DNA damage, and could be detected as foci formation in the nucleus (Dimitrova et al., 2008).
3. Cerebellar interneurons are not rescued inthe Xrcc1nes-cre; Atm-/-brain
To analyze the function of ATMformaintenance of genomic stability during brain development,we had surveyed the Xrcc1nes-cre; Atm-/- mouse brain using various markers of differentiated neural cells. In the previous study, Xrcc1nes-crebrains showed profound and widespread loss of cerebellar interneurons in the white matter (Lee et al., 2009). Interneuron progenitors of the Xrcc1nes-crecerebellumunderwent DNA damage induced p53 dependent cell cycle arrest rather than apoptosis resulting in absence of interneurons. To confirm this, theXrcc1nes-cre; p53-/-mouse had been generated and rescue of interneuron loss in the cerebellum was observed. p53 wasidentified as the first substrate of ATM in vitro and in vivo(Banin et al., 1998; Canman et al., 1998; Khanna et al., 1998).DNA damage triggers several signal transduction pathways that lead either to damage repair coupled with attenuation of cell cycle arrest, or to programmed cell death (apoptosis) (Choi et al., 2012;
Sullivan et al., 2012). Upon DNA damage, p53 is stabilized and activated through various posttranslational modifications including phosphorylation by ATM(Banin et al., 1998).
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Figure 3. The Xrcc1Nes-Cre;Atm-/-deficiency accumulates DNA damage during brain development. (A-B) The embryonic day 15.5 (E15.5) cerebellum (A) and cerebral cortex (CTX) (B) from Xrcc1Nes-Creand Xrcc1Nes-Cre;Atm-/- mice. Endogenous DNA damage was accumulated more in proliferating cells of the Xrcc1Nes-Cre;Atm-/-developing brain, as shown by γH2AX and 53BP1 foci formation in the nucleus. Nuclei were stained with DAPI. VZ,
Figure 3. The Xrcc1Nes-Cre;Atm-/-deficiency accumulates DNA damage during brain development. (A-B) The embryonic day 15.5 (E15.5) cerebellum (A) and cerebral cortex (CTX) (B) from Xrcc1Nes-Creand Xrcc1Nes-Cre;Atm-/- mice. Endogenous DNA damage was accumulated more in proliferating cells of the Xrcc1Nes-Cre;Atm-/-developing brain, as shown by γH2AX and 53BP1 foci formation in the nucleus. Nuclei were stained with DAPI. VZ,