Abstract
DNA double-strand breaks (DSBs) frequently occur in rapidly dividing cells such as proliferating progenitors during central nervous system development. If they cannot be repaired, these lesions will cause cell death. The non-homologous end joining (NHEJ) DNA repair pathway is the only pathway available to repair DSBs in post-mitotic neurons. The non-homologous end joining factor 1 (Nhej1) protein is a key component of the NHEJ pathway. Nhej1 interacts with Xrcc4 and Lig4 to repair DSBs. Loss of function of Xrcc4 or Lig4 is embryonic lethal in the mouse while the loss of Nhej1 is not. Surprisingly, the brains of Nhej1-deficient mice appear to be normal although NHEJ1 deficiency in humans causes severe neurological dysfunction and microcephaly. Here, we studied the consequences of Nhej1 dysfunction for the development of the cerebral cortex using in utero electroporation of inactivating small hairpin RNAs (shRNAs) in the developing rat brain. We found that decreasing Nhej1 expression during neuronal migration phases causes severe neuronal migration defects visualized at embryonic stages by an accumulation of heterotopic neurons in the intermediate zone. Knocked-down cells die by 7 days after birth and the brain regions where RNA interference was achieved are structurally abnormal, suffering from a reduction of the width of the external cortical layers. These results indicate that the Nhej1 protein is necessary for proper rat cortical development. Neurons unable to properly repair DNA DSBs are unable to reach their final destination during the development and undergo apoptosis, leading to an abnormal cortical development.
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Abbreviations
- CP:
-
Cortical plate
- DSBs:
-
Double-strand breaks
- HR:
-
Homologous recombination
- IZ:
-
Intermediate zone
- LIG4:
-
Ligase IV
- LV:
-
Lateral ventricle
- NeuN:
-
Neuronal nuclear antigen
- NHEJ:
-
Non-homologous end joining
- NHEJ1:
-
Non-homologous end joining factor 1
- TUNEL:
-
Terminal deoxynucleotidyltransferase-mediated dUTP end labeling
- VZ:
-
Ventricular zone
References
Symington LS, Gautier J (2011) Double-strand break end resection and repair pathway choice. Annu Rev Genet 45:247–271
Gao Y, Sun Y, Frank KM, Dikkes P, Fujiwara Y, Seidl KJ, Sekiguchi JM, Rathbun GA, Swat W, Wang J, Bronson RT, Malynn BA, Bryans M, Zhu C, Chaudhuri J, Davidson L, Ferrini R, Stamato T, Orkin SH, Greenberg ME, Alt FW (1998) A critical role for DNA end-joining proteins in both lymphogenesis and neurogenesis. Cell 95:891–902
Osborn AJ, Elledge SJ, Zou L (2002) Checking on the fork: the DNA-replication stress-response pathway. Trends Cell Biol 12:509–516
Gatz SA, Ju L, Gruber R, Hoffmann E, Carr AM, Wang ZQ, Liu C, Jeggo PA (2011) Requirement for DNA ligase IV during embryonic neuronal development. J Neurosci 31:10088–10100
Vyjayanti VN, Rao KS (2006) DNA double strand break repair in brain: reduced NHEJ activity in aging rat neurons. Neurosci Lett 393:18–22
Orii KE, Lee Y, Kondo N, McKinnon PJ (2006) Selective utilization of nonhomologous end-joining and homologous recombination DNA repair pathways during nervous system development. Proc Natl Acad Sci U S A 103:10017–10022
Ahnesorg P, Smith P, Jackson SP (2006) XLF interacts with the XRCC4-DNA ligase IV complex to promote DNA nonhomologous end-joining. Cell 124:301–313
Hammel M, Yu Y, Fang S, Lees-Miller SP, Tainer JA (2010) XLF regulates filament architecture of the XRCC4.ligase IV complex. Structure 18:1431–1442
Lieber MR (2010) The mechanism of double-strand DNA break repair by the nonhomologous DNA end-joining pathway. Annu Rev Biochem 79:181–211
McKinnon PJ (2009) DNA repair deficiency and neurological disease. Nat Rev Neurosci 10:100–112
O’Driscoll M, Cerosaletti KM, Girard PM, Dai Y, Stumm M, Kysela B, Hirsch B, Gennery A, Palmer SE, Seidel J, Gatti RA, Varon R, Oettinger MA, Neitzel H, Jeggo PA, Concannon P (2001) DNA ligase IV mutations identified in patients exhibiting developmental delay and immunodeficiency. Mol Cell 8:1175–1185
Buck D, Malivert L, de Chasseval R, Barraud A, Fondaneche MC, Sanal O, Plebani A, Stephan JL, Hufnagel M, le Deist F, Fischer A, Durandy A, de Villartay JP, Revy P (2006) Cernunnos, a novel nonhomologous end-joining factor, is mutated in human immunodeficiency with microcephaly. Cell 124:287–299
Cantagrel V, Lossi AM, Lisgo S, Missirian C, Borges A, Philip N, Fernandez C, Cardoso C, Figarella-Branger D, Moncla A, Lindsay S, Dobyns WB, Villard L (2007) Truncation of NHEJ1 in a patient with polymicrogyria. Hum Mutat 28:356–364
Barnes DE, Stamp G, Rosewell I, Denzel A, Lindahl T (1998) Targeted disruption of the gene encoding DNA ligase IV leads to lethality in embryonic mice. Curr Biol 8:1395–1398
Gu Y, Sekiguchi J, Gao Y, Dikkes P, Frank K, Ferguson D, Hasty P, Chun J, Alt FW (2000) Defective embryonic neurogenesis in Ku-deficient but not DNA-dependent protein kinase catalytic subunit-deficient mice. Proc Natl Acad Sci U S A 97:2668–2673
Vemuri MC, Schiller E, Naegele JR (2001) Elevated DNA double strand breaks and apoptosis in the CNS of scid mutant mice. Cell Death Differ 8:245–255
Li G, Alt FW, Cheng HL, Brush JW, Goff PH, Murphy MM, Franco S, Zhang Y, Zha S (2008) Lymphocyte-specific compensation for XLF/cernunnos end-joining functions in V(D)J recombination. Mol Cell 31:631–640
Buck D, Moshous D, de Chasseval R, Ma Y, le Deist F, Cavazzana-Calvo M, Fischer A, Casanova JL, Lieber MR, de Villartay JP (2006) Severe combined immunodeficiency and microcephaly in siblings with hypomorphic mutations in DNA ligase IV. Eur J Immunol 36:224–235
Nieto M, Monuki ES, Tang H, Imitola J, Haubst N, Khoury SJ, Cunningham J, Gotz M, Walsh CA (2004) Expression of Cux-1 and Cux-2 in the subventricular zone and upper layers II-IV of the cerebral cortex. J Comp Neurol 479:168–180
Sharma M, Brantley JG, Vassmer D, Chaturvedi G, Baas J, Vanden Heuvel GB (2009) The homeodomain protein Cux1 interacts with Grg4 to repress p27 kip1 expression during kidney development. Gene 439:87–94
Le Douce V, Colin L, Redel L, Cherrier T, Herbein G, Aunis D, Rohr O, Van Lint C, Schwartz C (2012) LSD1 cooperates with CTIP2 to promote HIV-1 transcriptional silencing. Nucleic Acids Res 40:1904–1915
Mullen RJ, Buck CR, Smith AM (1992) NeuN, a neuronal specific nuclear protein in vertebrates. Development 116:201–211
Cheong JW, Chong SY, Kim JY, Eom JI, Jeung HK, Maeng HY, Lee ST, Min YH (2003) Induction of apoptosis by apicidin, a histone deacetylase inhibitor, via the activation of mitochondria-dependent caspase cascades in human Bcr-Abl-positive leukemia cells. Clin Cancer Res 9:5018–5027
French CA, Groszer M, Preece C, Coupe AM, Rajewsky K, Fisher SE (2007) Generation of mice with a conditional Foxp2 null allele. Genesis 45:40–46
Rice H, Suth S, Cavanaugh W, Bai J, Young-Pearse TL (2010) In utero electroporation followed by primary neuronal culture for studying gene function in subset of cortical neurons. J Vis Exp Oct 8; (44). doi: 10.3791/2103
Cook PJ, Ju BG, Telese F, Wang X, Glass CK, Rosenfeld MG (2009) Tyrosine dephosphorylation of H2AX modulates apoptosis and survival decisions. Nature 458:591–596
Charles I, Khalyfa A, Kumar DM, Krishnamoorthy RR, Roque RS, Cooper N, Agarwal N (2005) Serum deprivation induces apoptotic cell death of transformed rat retinal ganglion cells via mitochondrial signaling pathways. Invest Ophthalmol Vis Sci 46:1330–1338
Corbo JC, Deuel TA, Long JM, LaPorte P, Tsai E, Wynshaw-Boris A, Walsh CA (2002) Doublecortin is required in mice for lamination of the hippocampus but not the néocortex. J Neurosci 22:7548–7557
Bai J, Ramos RL, Ackman JB, Thomas AM, Lee RV, LoTurco JJ (2003) RNAi reveals doublecortin is required for radial migration in rat neocortex. Nat Neurosci 6:1277–1283
Jackson SP, Bartek J (2009) The DNA-damage response in human biology and disease. Nature 461:1071–1078
Rakic P (2009) Evolution of the neocortex: a perspective from developmental biology. Nat Rev Neurosci 10:724–735
Lizarraga SB, Margossian SP, Harris MH, Campagna DR, Han AP, Blevins S, Mudbhary R, Barker JE, Walsh CA, Fleming MD (2010) Cdk5rap2 regulates centrosome function and chromosome segregation in neuronal progenitors. Development 137:1907–1917
Franco SJ, Gil-Sanz C, Martinez-Garay I, Espinosa A, Harkins-Perry SR, Ramos C, Müller U (2012) Fate-restricted neural progenitors in the mammalian cerebral cortex. Science 337:746–749
Barkovich AJ, Kuzniecky RI, Jackson GD, Guerrini R, Dobyns WB (2001) Classification system for malformations of cortical development: update 2001. Neurology 57:2168–2178
Abner CW, McKinnon PJ (2004) The DNA double-strand break response in the nervous system. DNA Repair (Amst) 3:1141–1147
Frank KM, Sharpless NE, Gao Y, Sekiguchi JM, Ferguson DO, Zhu C, Manis JP, Horner J, DePinho RA, Alt FW (2000) DNA ligase IV deficiency in mice leads to defective neurogenesis and embryonic lethality via the p53 pathway. Mol Cell 5:993–1002
Haydar TF, Kuan CY, Flavell RA, Rakic P (1999) The role of cell death in regulating the size and shape of the mammalian forebrain. Cereb Cortex 9:621–626
Acknowledgments
This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), Programme Hospitalier de Recherche Clinique (PHRC) from the French Ministry of Health and GIS Institut des Maladies Rares.
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The authors declare that they have no conflict of interest.
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El Waly, B., Buhler, E., Haddad, MR. et al. Nhej1 Deficiency Causes Abnormal Development of the Cerebral Cortex. Mol Neurobiol 52, 771–782 (2015). https://doi.org/10.1007/s12035-014-8919-y
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DOI: https://doi.org/10.1007/s12035-014-8919-y