Terminally differentiated astrocytes lack DNA damage response signaling and are radioresistant but retain DNA repair proficiency

Abstract

The impact and consequences of damage generation into genomic DNA, especially in the form of DNA double-strand breaks, and of the DNA-damage response (DDR) pathways that are promptly activated, have been elucidated in great detail. Most of this research, however, has been performed on proliferating, often cancerous, cell lines. In a mammalian body, the majority of cells are terminally differentiated (TD), and derives from a small pool of self-renewing somatic stem cells. Here, we comparatively studied DDR signaling and radiosensitivity in neural stem cells (NSC) and their TD-descendants, astrocytes - the predominant cells in the mammalian brain. Astrocytes have important roles in brain physiology, development and plasticity. We discovered that NSC activate canonical DDR upon exposure to ionizing radiation. Strikingly, astrocytes proved radioresistant, lacked functional DDR signaling, with key DDR genes such as ATM being repressed at the transcriptional level. Nevertheless, astrocytes retain the expression of non-homologous end-joining (NHEJ) genes and indeed they are DNA repair proficient. Unlike in NSC, in astrocytes DNA-PK seems to be the PI3K-like protein kinase responsible for γH2AX signal generation upon DNA damage. We also demonstrate the lack of functional DDR signaling activation in vivo in astrocytes of irradiated adult mouse brains, although adjacent neurons activate the DDR.

Figure 1

NSC show efficient DDR activation whereas this is strongly impaired in NSC-derived astrocytes. (a) At 1 h after irradiation with 10 Gy, NSC (here, negative for intermediate filament GFAP) uniformly display ATM kinase activity as demonstrated by the detection of foci of autophosphorylated ATM (S1981), ATM/ATR/DNA-PK-specific phospho-epitopes (pS/TQ), 53BP1 and of the phosphorylated histone H2AX (γH2AX), as analyzed by confocal microscopy of immunofluorescence stainings. Bar: 15 μm. (b) In contrast, astrocytes (positive for intermediate filament GFAP) when irradiated and treated in parallel with NSC, show only marginal phospho-ATM foci appearance and nearly no nuclear signal of 53BP1 or pS/TQ foci. However, astrocytes still display irradiation-induced nuclear γH2AX signal. Bar: 15 μm

Figure 2

DDR response factors are transcriptionally suppressed in astrocytes, whereas NSC show canonical DDR upon irradiation. (a) Quantitative RT-PCR analysis reveals a transcriptional downregulation of DDR genes, but retained expression of DNA repair factors, in astrocytes. Expression profiles were normalized against parental NSC before serum-induced differentiation. β2-microglobulin was used as a housekeeping gene. (b) Western blot analysis of NSC and astrocytes, irradiated and processed in parallel. Membranes were probed with phospho-specific and total antibodies as shown and normalized for vinculin. (c) Quantitative RT-PCR analysis showing that even 24 h following irradiation, key DDR factors remain downregulated in astrocytes. Four target genes of p53 were also analyzed: transcripts of GADD45a, BAX and PUMA remained largely unchanged. The mRNA of the cell-cycle control gene p21^CIP was found upregulated. Expression profiles were normalized against non-irradiated astrocytes. β2-microglobulin was used as a housekeeping gene

Figure 3

Residual ATM activity can be detected in irradiated astrocytes and parallels the downregulation of the γH2AX signal. (a) Western blot analysis of DDR kinetics in astrocytes, irradiated with 10 Gy. Weak and transient phospho-ATM signal can be detected and coincides with reduction of γH2AX and increase in p53 signal. Note that even 50 Gy fail to induce any strong DDR in astrocytes. Irradiated NSC were used as positive control. Membranes were probed with phospho-specific and total antibodies and normalized for vinculin. (b) TUNEL assay for apoptosis-induced DSB in astrocytes 72 h after irradiation with 10 Gy. Note that also re-irradiation with further 10 Gy 24 h after the first exposure to X-rays fails to induce apoptosis. NSC 72 h after 10 Gy irradiation show a profound induction of apoptosis compared with non-irradiated NSC. (c) Immunofluorescence analysis showing the DNA-damage-induced appearance of γH2AX signal in irradiated astrocytes. After 24 h, the foci are strongly downregulated, indicating repair. Another round of irradiation leads to a de novo formation of the γH2AX signal, indicating a still functional H2AX phosphorylation machinery. Bar: ∼22 μm. (d) Similar observations can also be made by western blot analysis. Membrane was probed with phospho-specific and total antibody against H2AX and normalized for α-tubulin

Figure 4

Astrocytes display functional ATM-biased DNA repair and DNA-PK-biased H2AX phosphorylation, but no apoptotic response upon irradiation. (a) Western blot analysis of NSC and astrocytes, treated with a solvent (DMSO) or DNA-PK inhibitor NU7441 and ATM inhibitor KU55933, separately or in combination (NU+KU) at concentrations of 1 μM each (lower than IC50 values of the inhibitor's second most sensitive PI3K-like protein kinase target), irradiated and processed in parallel. The membrane was probed for the phosphorylated and total form of H2AX and normalized with vinculin. (b) Flow cytometrical analysis of astrocytes, treated with inhibitors as above, irradiated and stained with antibody against γH2AX. Alexa488 secondary antibody signal was measured on log₁₀ scale. Gates were set to discriminate γH2AX-negative cells, whereas γH2AX positive cells were arbitrarily subdivided into high and low positive. Same gateset was used for all measurements of each experiment done in quadruplicate. Error bars show S.D. (c) Confocal immunofluorescence analysis of astrocytes irradiated with 1 Gy to better discriminate γH2AX foci. Note the reduction of the γH2AX signal intensity in irradiated astrocytes, treated with DNA-PK inhibitor NU7441. Bar: 10 μm. (d) DSB detection by neutral COMET assay in astrocytes 1 and 24 h after irradiation, treated with inhibitors as above. Olive tail moments are presented as box plot diagram, with vertical bars indicating median values. Error bars show S.D. Significance of the median values was calculated with Dunn's method with DMSO-treated cells as control group. ^‡same significance ratio applies when non-irradiated cells are used as reference. (e) TUNEL assay for apoptosis-induced DSB in astrocytes 24 and 72 h after irradiation, treated with inhibitors as above. As a positive control for apoptotic proficiency of astrocytes, cells were treated for 2 days with puromycin (5 μg/ml) or hygromycin (800 μg/ml)

Figure 5

NSC-derived neurons are DDR-proficient. Also in vivo neurons show robust DDR upon irradiation, as opposed to brain astrocytes. (a) Terminally differentiated neurons were derived from NSC according to an established differentiation protocol. At 1 h after irradiation with 10 Gy both NSC (here, negative for neuronal marker Tuj1) and NSC-derived neurons (Tuj1 positive) show robust ATM kinase activity through the appearance of foci of phospho-epitope (pS/TQ) as analyzed by confocal immunofluorescence. Bar: 10 μm. (b) Wild-type mice were untreated or irradiated with a sub-lethal dose of 8 Gy and killed for brain analysis after 6 h. Neurons were detected using nuclear neuron marker NeuN and astrocytes using intermediate filament GFAP. Activation of DDR was assessed through formation of 53BP1 foci upon irradiation. Neurons show 53BP1 foci assembly 6 h after 8 Gy delivery, whereas astrocytes do not. Note that astrocytes also lack the diffuse nuclear 53BP1 signal present in neurons. Bar: 10 μm. Note: GFAP protein in brain astrocytes is known to localize only in their cellular protrusions, hence different signal appearance from GFAP of in vitro astrocytes