Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2000 Jan 18;97(2):889-94.
doi: 10.1073/pnas.97.2.889.

Atm and Bax cooperate in ionizing radiation-induced apoptosis in the central nervous system

M J Chong  1 , M R Murray, E C Gosink, H R Russell, A Srinivasan, M Kapsetaki, S J Korsmeyer, P J McKinnon
Affiliations

Atm and Bax cooperate in ionizing radiation-induced apoptosis in the central nervous system

M J Chong et al. Proc Natl Acad Sci U S A. .

Abstract

Ataxia-telangiectasia is a hereditary multisystemic disease resulting from mutations of ataxia telangiectasia, mutated (ATM) and is characterized by neurodegeneration, cancer, immune defects, and hypersensitivity to ionizing radiation. The molecular details of ATM function in the nervous system are unclear, although the neurological lesion in ataxia-telangiectasia becomes apparent early in life, suggesting a developmental origin. The central nervous system (CNS) of Atm-null mice shows a pronounced defect in apoptosis induced by genotoxic stress, suggesting ATM functions to eliminate neurons with excessive genomic damage. Here, we report that the death effector Bax is required for a large proportion of Atm-dependent apoptosis in the developing CNS after ionizing radiation (IR). Although many of the same regions of the CNS in both Bax-/- and Atm-/- mice were radioresistant, mice nullizygous for both Bax and Atm showed additional reduction in IR-induced apoptosis in the CNS. Therefore, although the major IR-induced apoptotic pathway in the CNS requires Atm and Bax, a p53-dependent collateral pathway exists that has both Atm- and Bax-independent branches. Further, Atm- and Bax-dependent apoptosis in the CNS also required caspase-3 activation. These data implicate Bax and caspase-3 as death effectors in neurodegenerative pathways.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Bax is required for IR-induced apoptosis in the cerebellum and dentate gyrus. (A) Apoptosis is almost absent in the P5 Bax−/− dentate gyrus (c and d) compared with WT tissue (a and b) 18 hr after IR. Apoptosis was assessed by neutral red (a and c) and Sytox green (b and d). (B) Apoptosis occurs in the EGL of the P5 WT cerebellum (a and e) but is markedly reduced or absent in Atm−/− (b and f), p53−/− (c and g), and Bax−/− (d and h) tissue after IR. In contrast to in the cerebellum, apoptosis in the central region of the WT (i) and Bax −/− retina (k) are indistinguishable, whereas in the Atm−/− retina (j), it is absent. a–d are neutral red stained, and e–k are in situ end labeling-stained tissue 18 hr after IR. (×200.)
Figure 2
Figure 2
p53 stabilization occurs in Bax−/− cerebellum, whereas Bax protein levels are unaffected by IR. (A) Examination of p53 stabilization by using immunohistochemistry shows equivalent p53 stabilization in the EGL of WT and Bax−/− cerebellum after IR but reduced stabilization in Atm−/− and an absence in p53−/− tissue. (×200.) (B) Bax levels do not alter up to 5 hr after IR. After identification of p53, the immunoblot was reprobed with an antibody to Bax; 1–4 are P5 cerebellum samples 0, 0.5, 1, and 5 hr after IR, respectively. (C) p53 is induced in WT and Bax−/− eye after IR (lanes 5 and 8, respectively), whereas induction is reduced in Atm−/− and absent from p53−/− tissue (lanes 6 and 7, respectively). The induction of p53 in the Atm−/− retina occurs in multipotential cells of the periphery, where IR-induced apoptosis is independent of Atm (4). Bax levels are similar in WT, Atm−/−, and in p53−/− cerebellum and eye (1–3, respectively) and are not obviously affected by IR (WT; Atm and p53 are 5, 6, and 7, respectively). Bax−/− tissues (4 and 8) have no detectable Bax. Lanes 1–4 and 5–8 are extracts from unirradiated or irradiated animals, respectively. Control is a region of the Ponceau S-stained membrane used for the anti-Bax immunoblot of the cerebellum to illustrate protein loading and transfer.
Figure 3
Figure 3
Apoptosis is further reduced in Atm/Bax double-null CNS tissue after IR. (A) Sytox green staining of the P5 cerebellum from WT, Atm-null, and Atm/Bax double-null animals shows a pronounced reduction in the number of apoptotic EGL cells 18 hr after IR in the Atm-null compared with WT. A further reduction was seen in Atm/Bax double-null EGL. Arrows in the Atm−/− section indicate apoptotic cells. (×400.) (B) Quantitative comparison of irradiated cerebellum from various genotypes scored for apoptotic cells. Although the WT cerebellum had abundant cell deaths, the Atm-null and Bax-null cerebellum showed only low numbers of dead cells, whereas the Atm/Bax double-null cerebellum was indistinguishable from the p53-null cerebellum, with an almost complete absence of apoptosis.
Figure 4
Figure 4
Caspase-3 activation coincides with apoptosis in the CNS of WT mice. (A) Apoptotic cells appeared between 2 and 3 hr in the cerebellar EGL after IR and by 4 hr, apoptotic figures were widely distributed throughout the EGL. Sections are stained with Sytox green to visualize pyknotic cells; arrows indicate apoptotic cells. (×400.) Immunohistochemistry by using antibodies specific for the cleaved subunit of caspase-3 (15) show caspase-3 activation precedes the apoptotic morphological changes. Caspase-3 cleavage appears initially at 2 hr in the outer EGL, and abundant staining throughout the EGL occurs at 3 and 4 hr. (×200.) (B) Caspase-3 enzyme activity coincides with caspase-3 cleavage and apoptosis seen in A. An increase in caspase-3 activity (pmol of 7-amino-4-methylcoumarin liberated per μg of protein) occurred between 2 and 4 hr after IR in the cerebellum. Caspase-1 activity was insensitive to radiation. (C) Cerebellar slice cultures were irradiated, incubated with various caspase inhibitors, cryosectioned, and stained with neutral red to identify apoptotic cells. Incubation with general caspase inhibitors (z-VAD-FMK) or caspase-3 inhibitor (z-DVED-FMK), but not caspase-1 inhibitor (z-YVAD-CHO) or DMSO vehicle alone (CONTROL), prevented IR-induced apoptosis in the cerebellar EGL. No apoptosis was observed in unirradiated cultures. Sections are from P5 cerebellum. (×400.)
Figure 5
Figure 5
Caspase expression and proteolysis after IR. (A) RPA analysis of the P5 WT, Atm, p53, and Bax-null cerebellum shows a high level of expression for caspase-3 and only low levels for caspases-2, -6, and -8. (B) Protein immunoblot analysis with anticaspase-3 shows equal levels of caspase-3 proenzyme are present in WT, Atm−/−, p53−/−, and Bax−/− tissues. (C) Caspase-3 activation was reduced in the Atm- and Bax-null cerebellar EGL and further reduced in the Atm/Bax double-null animals after irradiation. No caspase-3 activation was observed in the irradiated p53-null cerebellum. (×200.) (D) The relative levels of caspase-3 activity in various mutant tissues 6 hr after IR as a percentage of WT activity. In all cases, samples were normalized for caspase-3 activity present in unirradiated tissues. DG, dentate gyrus.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Sedgwick R P, Boder E. In: Handbook of Clinical Neurology. Vinken P, Bruyn G, Klawans H, editors. Vol. 60. New York: Elsevier; 1991. pp. 347–423.
    1. Lavin M F, Shiloh Y. Annu Rev Immunol. 1997;15:177–202. - PubMed
    1. Soares H D, Morgan J I, McKinnon P J. Neuroscience. 1998;86:1045–1054. - PubMed
    1. Herzog K H, Chong M J, Kapsetaki M, Morgan J I, McKinnon P J. Science. 1998;280:1089–1091. - PubMed
    1. Adams J M, Cory S. Science. 1998;281:1322–1326. - PubMed

Publication types

MeSH terms