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Polynucleotide kinase-phosphatase enables neurogenesis via multiple DNA repair pathways to maintain genome stability - PubMed

  • ️Thu Jan 01 2015

Polynucleotide kinase-phosphatase enables neurogenesis via multiple DNA repair pathways to maintain genome stability

Mikio Shimada et al. EMBO J. 2015.

Abstract

Polynucleotide kinase-phosphatase (PNKP) is a DNA repair factor possessing both 5'-kinase and 3'-phosphatase activities to modify ends of a DNA break prior to ligation. Recently, decreased PNKP levels were identified as the cause of severe neuropathology present in the human microcephaly with seizures (MCSZ) syndrome. Utilizing novel murine Pnkp alleles that attenuate expression and a T424GfsX48 frame-shift allele identified in MCSZ individuals, we determined how PNKP inactivation impacts neurogenesis. Mice with PNKP inactivation in neural progenitors manifest neurodevelopmental abnormalities and postnatal death. This severe phenotype involved defective base excision repair and non-homologous end-joining, pathways required for repair of both DNA single- and double-strand breaks. Although mice homozygous for the T424GfsX48 allele were lethal embryonically, attenuated PNKP levels (akin to MCSZ) showed general neurodevelopmental defects, including microcephaly, indicating a critical developmental PNKP threshold. Directed postnatal neural inactivation of PNKP affected specific subpopulations including oligodendrocytes, indicating a broad requirement for genome maintenance, both during and after neurogenesis. These data illuminate the basis for selective neural vulnerability in DNA repair deficiency disease.

Keywords: DNA repair; neurodevelopment; neurologic disease; polynucleotide kinase–phosphatase.

© 2015 The Authors.

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Figures

Figure 1
Figure 1

PNKP is essential factor in neurogenesis

  1. A A conditional Pnkp allele (cKO) was generated by flanking exons 4 through 7 with LoxP sites. Cre-mediated excision generated a transcript that joined exons 3 and 8, thereby changing the Pnkp reading frame to a premature stop codon leading to a truncated protein.

  2. B Western blot analysis showed that PnkpNes-cre mice underwent inactivation of PNKP in neural tissues (the cortex) but not tissues outside of the nervous system (the liver). NBS1 is used as an internal standard, and Ponceau staining indicates equivalent protein transfer.

  3. C PnkpNes-cre mice were born alive, but failed to survive past 5 days of age.

  4. D Loss of PNKP markedly affected development of the brain as reduction in the size of the cortex (red-hatched lines) and cerebellum is observed in brain photographs and Nissl-stained sagittal sections.

  5. E While PNKP loss affected brain development by a reduction in overall cell number, the ordered six-layer lamination present in the cortex is nonetheless still maintained in the mutant (indicated by roman numerals).

Figure 2
Figure 2

PNKP prevents DNA damage-induced apoptosis in the developing nervous system

  1. A TUNEL (green) with propidium iodide (PI, red) counterstaining identifies abundant apoptosis in the E15.5 developing nervous system of the PnkpNes-cre embryo compared to the base excision repair mutant Xrcc1Nes-cre and the NHEJ mutant Lig4Nes-cre or wild-type controls. TUNEL-positive cells are quantified in the graphs opposite. ***P < 0.0003.

  2. B Loss of PNKP resulted in DNA damage as indicated by γH2AX immunostaining, both as small nuclear puncta indicating DNA damage and also as large diffuse apoptotic foci. Xrcc1Nes-cre and Lig4Nes-cre are shown as a comparison to PnkpNes-cre. Quantitative data are shown on adjacent graphs. PCNA immunostaining was used to identify proliferating cells.

  3. C BrdU incorporation shows there is a marked reduction in cell proliferation in the PnkpNes-cre embryonic tissue compared to either Xrcc1Nes-cre or Lig4Nes-cre. Adjacent graphs are quantitative representation of the immunostains. ***P < 0.005.

Data information: For (A–C) the mean (± SD) results from at least 3 replicates.

Figure 3
Figure 3

PNKP loss targets multiple cell types and involves p53 signaling

  1. A PNKP loss results in apoptosis in the developing cerebellum as shown using TUNEL (green) with propidium iodide (PI, red) counterstaining. Apoptosis is substantially rescued by p53 inactivation but not ATM inactivation. Arrows indicate apoptotic cells.

  2. B Cell loss after inactivation of PNKP involves p53, but not ATM, as illustrated by rescue of upper layer neurons in (Pnkp;p53)Nes-cre tissue (identified by Satb2 immunostaining); asterisks indicate relative cortical rescue of PnkpNes-cre after p53 loss. Graphs quantify rescue after p53 loss. The mean (± SD) results from 3 independent sections. **P < 0.006.

  3. C Cerebellar interneurons (identified by Pax2 immunostaining) are also susceptible to PNKP loss and occur in a partially p53 manner. Data is the mean value of triplicate sections, error bars represent ± SD. ***P < 0.001.

Figure 4
Figure 4

PNKP is essential for DNA repair by both base excision repair and non-homologous end-joining

  1. A Genotoxins that cause single- (IR, H2O2 and CPT) and double (IR)-strand breaks in non-replicating (density arrested) primary astrocytes show a repair deficiency after PNKP loss via the alkaline comet assay. IR is ionizing radiation, H2O2 is hydrogen peroxide and is used at 4°C to restrict DNA damage to single-strand breaks, and CPT is camptothecin. ***P < 0.0001, **P < 0.06.

  2. B Primary PnkpNes-cre astrocytes show a defect in the repair of DNA DSBs after bleomycin treatment when analyzed using the neutral comet assay system. The alkaline comet assay identifies both SSBs and DSBs while the neutral comet assay identifies DSBs. ***P < 0.0001; ns, not significant.

  3. C Recovery of the DSB markers, γH2AX and 53BP1, are defective in PnkpNes-cre astrocytes after IR as it is in the Lig4Nes-cre NHEJ mutant, while the Xrcc1Nes-cre base excision repair mutant is not defective in NHEJ. Quantitation of γH2AX removal is presented graphically.

  4. D PnkpNes-cre primary astrocytes also show frequent micronuclei formation after radiation, in a manner distinct to Lig4Nes-cre or Xrcc1Nes-cre. Chromosome bridges are also found in PnkpNes-cre astrocytes after radiation as shown in the photomicrograph.

Data information: For (A–D) the mean (± SD) results from at least 3 experimental replicates.

Figure 5
Figure 5

PNKP is dispensable for DNA repair by homologous recombination

  1. A Homologous recombination (HR) after mitomycin C (MMC) exposure was monitored in primary astrocytes lacking PNKP, BRCA2 or ATM by recovery of damage-induced γH2AX and RAD51 foci utilizing immunocytochemistry. In contrast to PNKP deficiency, cells deficient in the HR factor BRCA2 have a measurable deficit in DNA repair after MMC, while ATM-null cells do not show defective HR.

  2. B Quantitation of repair and recovery of γH2AX and RAD51 foci in (A) is shown graphically. The mean (± SD) is from at least 3 replicates. ***P < 0.003.

Figure 6
Figure 6

A hypomorphic germ line Pnkp allele with attenuated PNKP levels impacts development and DNA repair

  1. A A neomycin selection cassette (Neo) was inserted in intron 4 to generate the PnkpNeo allele.

  2. B Western blot analysis shows that the PnkNeo/Neo allele attenuates PNKP levels. Shorter and longer exposure show relative PNKP levels in the PnkpNeo/Neo tissue; the ratio of protein levels in the Pnkp mutant compared with controls is shown below the blots. Overall brain size is mildly affected compared to complete PNKP inactivation (arrows in photograph and Nissl-stained sagittal cryosections).

  3. C Attenuated PNKP level is associated with an increase in DNA damage formation (γH2AX) and apoptosis (TUNEL), and reduced immunostaining of the proliferation markers PCNA and pH3. Arrows indicate the basal and apical cortical progenitors. Adjacent graphs quantify DNA damage, apoptosis and proliferation. The mean (± SD) is from at least 3 replicates. **P < 0.009.

  4. D Quiescent primary mouse embryonic fibroblasts (MEFs) from PnkpNeo/Neo embryos also show a DNA repair defect after camptothecin (CPT) as determined using alkaline comet assays. Repair of ionizing radiation (IR) and methyl methanesulfonate (MMS) is also defective in PnkpNeo/Neo proliferating MEFs as determined using alkaline comet assays. Tdp1−/− and (Lig4;p53)−/− MEFs were also used as a positive control for defective DNA repair. The mean (± SD) is from at least 3 replicates. ***P < 0.0001, **P < 0.005.

Figure 7
Figure 7

Induced Pnkp deletion in the postnatal brain affects multiple cell types

  1. A Inducible Pnkp deletion commencing at postnatal day 21 (P21) using a tamoxifen (TX)-inducible actin-based promoter reveals little overall effect toward brain size or structure.

  2. B Western blot analysis shows reduced PNKP levels after CreTM induction in multiple brain regions. There is also a reduction of myelin basic protein (MBP) in the PnkpCreTM brain. Actin serves as a loading control.

  3. C Analysis of the PnkpCreTM brain after TX administration using the neuronal marker NeuN, which immunostains mature neurons, revealed a marked reduction of this marker in the dentate gyrus (DG) and the hippocampal CA3 region (red-hatched boxes), and the cerebellum. The myelin-producing oligodendrocytes also show a reduction in myelination as judged by reduced MBP immunostaining (arrows), consistent with reduced protein levels in (B). Arrowheads indicate that although NeuN immunostaining is decreased in the mutant, this is not due to cell loss as seen in the DAPI-stained merged image.

Figure 8
Figure 8

PNKP is an essential maintenance factor for glia in the postnatal brain after neurogenesis

  1. A Selective inactivation of Pnkp in the glia using GFAP-creTM reveals an important role for PNKP function in maintaining these neural cells. Pnkp deletion at P1 results in a brain with mild cortical and cerebellar size reduction (asterisk).

  2. B Myelin basic protein immunostaining is strongly reduced by P20 in the PnkpGFAP-creTM brain after induction at P1. NeuN (Fox-3) immunostaining also reveals alteration in nuclear morphology, likely as an indirect consequence of PNKP inactivation in glia.

  3. C PnkpGFAP-creTM broadly impacts oligodendrocytes as CNPase and Olig2 immunostaining are also reduced.

  4. D Alteration in MBP, CNPase and Olig2 does not involve loss of oligodendrocytes as revealed by normal numbers of Sox10-positive cells. Graph shows quantitation of oligodendrocyte cell bodies (Sox10) in the control and PnkpGFAPCreTM cortex. The mean (± SD) is from at least 3 sections. ns, not significant.

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