Analysis of the ataxia telangiectasia mutated-mediated DNA damage response in murine cerebellar neurons

Abstract

The DNA damage response is a network of signaling pathways that affects many aspects of cellular metabolism after the induction of DNA damage. The primary transducer of the cellular response to the double-strand break, a highly cytotoxic DNA lesion, is the nuclear protein kinase ataxia telangiectasia (A-T) mutated (ATM), which phosphorylates numerous effectors that play key roles in the damage response pathways. Loss or inactivation of ATM leads to A-T, an autosomal recessive disorder characterized by neuronal degeneration, particularly the loss of cerebellar granule and Purkinje cells, immunodeficiency, genomic instability, radiosensitivity, and cancer predisposition. The reason for the cerebellar degeneration in A-T is not clear. It has been ascribed by several investigators to cytoplasmic functions of ATM that may not be relevant to the DNA damage response. We set out to examine the subcellular localization of ATM and characterize the ATM-mediated damage response in mouse cerebellar neurons. We found that ATM is essentially nuclear in these cells and that various readouts of the ATM-mediated damage response are similar to those seen in commonly used cell lines. These include the autophosphorylation of ATM, which marks its activation, and phosphorylation of several of its downstream substrates. Importantly, all of these responses are detected in the nuclei of granule and Purkinje cells, suggesting that nuclear ATM functions in these cells similar to other cell types. These results support the notion that the cerebellar degeneration in A-T patients results from defective DNA damage response.

Figure 1.

ATM localization in cerebellar organotypic cultures. A, ATM in Purkinje neuron and in surrounding cells resides in the molecular layer of the cerebellum. In Purkinje neuron, the ATM immunoreactivity is located mainly in the nucleus (white arrow); however, cytoplasmic traces of ATM can be seen. The molecular layer contains several types of neurons such as the basket and satellite neurons. B, This image clearly demonstrates that ATM is predominantly nuclear in these cells. The cultures were fixed and reacted with anti-ATM antibodies (5C2). Scale bars, 20 μm.

Figure 2.

Atm activation after DNA damage. A, Total protein extracts were isolated from Atm^−/− and Atm^+/+ mice cerebellar granule neurons untreated and treated with NCS (200 ng/ml, 30 min), and immunoblots were reacted with anti-pS1981 ATM antibody (1:1000) and then with anti-ATM antibody (Mat3 [anti-ATM antibody that was prepared against a short sequence (peptide) of murine ATM], 1:1000). This monoclonal antibody was prepared against peptide derived from mouse Atm. To verify equal loading, the blot was stripped and reacted with anti-tubulin antibody. As expected, anti-ATM antibody immunoreacts only with extracts isolated from Atm^+/+ cerebella. Note that anti-pS1981 ATM antibody reacts only with activated Atm. B, Dose-dependent activation of Atm. Cerebellar organotypic cultures (day 12 in primary culture) were exposed to various doses of IR (0, 2, 5, and 10 Gy) for 30 min, fixed, and treated with anti-pS1981 ATM antibody (Bethyl Laboratories, Montgomery, TX). Purkinje cells were identified using anti calbindin-28 antibody (red). Note that only the nuclei of Atm^+/+ cerebellar neurons that were treated with IR are labeled, suggesting that DNA damage activates Atm in the nuclei of the various cerebellar cells and Purkinje cells. Left panels show the images of fluorescent anti-pS1981 ATM antibody (green), and the right panels are merged images of the fluorescent anti-pS1981 ATM antibody (green), anti-calbindin-28 antibody (red), and Sytox blue. C, Time course of Atm activation in response to IR. Cerebellar organotypic cultures were exposed to 10 Gy, and, at various time points after irradiation, the cultures were fixed and treated with anti-pS1981 ATM antibody. Purkinje cells were identified using anti calbindin-28 antibody (red). Note that Atm is activated as early as 5 min after irradiation and remains active for 2 h. D, Evidence at higher magnification of nuclear foci formed by activated ATM. Merge, α-pS1981–ATM plus calbindin-28 plus Sytox blue. Scale bars, 10 μm.

Figure 3.

Atm activation after exposure of whole mice to 20 Gy IR. Brain sections were isolated from untreated and irradiated 1-month-old (Atm^+/+ and Atm^−/−) mice. The brains were fixed, and frozen sections were prepared. The sections treated with anti-pS1981) ATM antibody (1:2000; Bethyl Laboratories). Purkinje cells were visualized using anti-calbindin-28 antibody. The panels show a merged picture [calbindin D28K antibody (green) and anti-pS1981 ATM antibody (red)]. Scale bar, 20 μm. PCL, Purkinje cell layer.

Figure 4.

Partial ATM dependence of γH2AX focus formation after DNA damage in cerebellar organotypic cultures. One of the early events in the DSB response is histone H2AX phosphorylation on Ser139 and foci formation of γH2AX at the region of the damage sites. Purkinje cells were identified using anti-calbindin-D28K antibody (green in A, red in C). A, Organotypic cultures were treated with various doses of IR, and, 30 min later, the cultures were fixed and reacted with an anti-γH2AX antibody. γH2AX (in red) can be seen in Atm^+/+ as well in Atm^−/− cells after treatment with various doses of IR. Merge, α-γH2AX plus calbindin-28 plus Sytox blue. B, Higher magnification of Atm^+/+ and Atm^−/− cultures that were irradiated with 10 Gy and fixed after 30 min show smaller size of foci in Purkinje cells in contrast to surrounding cells, which have distinct and large foci. Scale bar, 10 μm. C, Organotypic cultures were exposed to 10 Gy, and, at various time points after irradiation, the cultures were fixed and treated with anti-γH2AX antibody (green). γH2AX foci are formed in Atm^+/+ cells as early as 5 min after irradiation and last up to 2 h after damage. In contrast, Atm-deficient cells are able to efficiently form γH2AX foci only later (starting at 30 min). Scale bar, 10 μm.

Figure 5.

Colocalization of activated ATM and γH2AX in irradiated organotypic cultures. Cerebellar organotypic cultures prepared from Atm^+/+ mice were exposed to 10 Gy, and, at various time points thereafter, the cultures were fixed and cotreated with anti-pS1981 ATM antibody (Bethyl Laboratories) (red) and anti- γH2AX (green). The white spots represent colocalization of the two antibodies. Note that ATM is activated throughout the irradiated nucleus, not only at the damage sites. Scale bar, 20 μm.

Figure 6.

Phosphorylation of Atm substrates (SQ/TQ response) after DNA damage in organotypic culture. Cerebellar organotypic cultures (day 12 in primary culture) prepared from Atm^+/+ or Atm^−/− mice were exposed to ionizing radiation, fixed, and treated with anti-phosphorylated SQ™Q antibodies. Purkinje cells were identified using anti-calbindin-D28K antibody (red). Cerebellar organotypic cultures were exposed to 10 Gy, and, at various time points (15 and 60 min) after irradiation, the cultures were fixed and reacted with anti-phosphorylated SQ™Q antibodies. Scale bar, 20 μm. Strong nuclear localization of phosphorylated SQ™Q proteins develops as DNA damage increases. Merge, α-p-SQ/TQ plus calbindin28 plus Sytox blue.

Figure 7.

Phosphorylation of specific ATM substrates. A, p53 activation after DNA damage in cultured granule cells. After DNA damage, p53 is activated and stabilized as a result of several posttranslational modifications. Western blot analysis of cultured granule neuronal proteins extracted 30 min after NCS treatment shows that p53 is phosphorylated heavily at Ser18 only in Atm^+/+ cells. This phosphorylation lasts 4 h and then declines (data not shown). Western blot analysis of cerebellar protein extracts 30 min after irradiation show that pS957 SMC1 (B) after DNA damage seen only in irradiated Atm^+/+ cerebellum.