Caspase-activated DNase/DNA fragmentation factor 40 mediates apoptotic DNA fragmentation in transient cerebral ischemia and in neuronal cultures

Abstract

Nuclear changes, including internucleosomal DNA fragmentation, are characteristic features of neuronal apoptosis resulting from transient cerebral ischemia and related brain insults for which the molecular mechanism has not been elucidated. Recent studies suggest that a caspase-3-mediated mechanism may be involved in the process of nuclear degradation in ischemic neurons. In this study, we cloned from rat brain a homolog cDNA encoding caspase-activated deoxyribonuclease (CAD)/DNA fragmentation factor 40 (DFF40), a 40 kDa nuclear enzyme that is activated by caspase-3 and promotes apoptotic DNA degradation. Subsequently, we investigated the role of CAD/DFF40 in the induction of internucleosomal DNA fragmentation in the hippocampus in a rat model of transient global ischemia and in primary neuronal cultures under ischemia-like conditions. At 8-72 hr after ischemia, CAD/DFF40 mRNA and protein were induced in the degenerating hippocampal CA1 neurons. CAD/DFF40 formed a heterodimeric complex in the nucleus with its natural inhibitor CAD (ICAD) and was activated after ischemia in a delayed manner (>24 hr) by caspase-3, which translocated into the nucleus and cleaved ICAD. Furthermore, an induced CAD/DFF40 activity was detected in nuclear extracts in both in vivo and in vitro models, and the DNA degradation activity of CAD/DFF40 was inhibited by purified ICAD protein. These results strongly suggest that CAD/DFF40 is the endogenous endonuclease that mediates caspase-3-dependent internucleosomal DNA degradation and related nuclear alterations in ischemic neurons.

Fig. 1.

Cloning of rat CAD/DFF40. a, Deduced amino acid sequence of rat CAD/DFF40 (GenBank accession number AF 136598) and comparison of amino acid sequences among rat, mouse (GenBank accession number AB 009377), and human DFF40 (GenBank accession number AB 013918). Identical amino acids are presented asdashes. The rat sequence contains a nuclear localization segment at its C terminus (bold andunderlined). b, Cotransfection of rat CAD/DFF40 and ICAD, but not CAD/DFF40 or ICAD alone, enhances STS-induced internucleosomal DNA fragmentation in human293 cells. DNA extraction and gel electrophoresis were performed 6 hr after STS treatment (1 μm).

Fig. 2.

Northern blot analysis of CAD/DFF40 mRNA in the rat. Total RNA was isolated from rat tissues and electrophoresed on a 1% agarose–formaldehyde gel (30 μg of RNA per lane). The only transcription species resulting from hybridizing with the CAD/DFF40 cDNA probe is ∼2.7 kb. a, Distribution of CAD/DFF40 mRNA in various adult rat tissues. b, Regulation of CAD/DFF40 mRNA expression in the cerebellum during development. 17-day E, Embryonic day 17; 1-week p, postnatal 1 week;2-week p, postnatal 2 weeks. In all Northern blot analyses, the same blot was hybridized with the GADPH probe to serve as a control for sample loading. The graphs under theblots illustrate the relative levels of CAD/DFF40 mRNA expression in tissues, determined by optical density measurement on autoradiograms from two independent experiments. All densitometric values for CAD/DFF40 were normalized to that for GAPDH determined on the same lane.

Fig. 3.

Alterations of CAD/DFF40 mRNA expression after cerebral ischemia. a, Top, Northern blot analysis of CAD/DFF40 mRNA in the hippocampus after sham operation or 8, 24, or 72 hr after ischemia. Total RNA was isolated from the hippocampi (three brains per time point) and electrophoresed through a 1% agarose–formaldehyde gel (30 μg of RNA per lane).Bottom, the same blot hybridized with the GAPDH probe serves as the sample-loading control. b, In situ hybridization analysis of CAD/DFF40 mRNA expression in the hippocampus after ischemia or sham operation. The graph illustrates the relative CAD/DFF40 mRNA changes in the hippocampal CA1 sector, CA3 sector, and dentate gyrus (DG) at 4, 8, 24, and 72 hr after ischemia versus sham controls (n = 4 per time point), determined by optical density measurement on autoradiograms. Data are mean ± SEM and represent percentage changes in ischemic brains versus sham controls. *p < 0.05 versus sham controls (ANOVA and post hoc Scheffe's tests). c, Representative emulsion-coated sections counterstained with TUNEL from a brain 72 hr after ischemia (B, D, E) and a sham control brain (A, C). Note that increased silver grains localize to TUNEL-positive (yellow stains) CA1 pyramidal neurons (B) and neurons in the caudate putamen (D, E). Magnification, 400×.

Fig. 4.

Alterations in CAD/DFF40 protein expression after cerebral ischemia. a, Western blot analysis of CAD/DFF40 in the nuclear (N) and cytosolic (C) fractions prepared from normal brain cells (Brain) and primary cortical neuron cultures (Neuron). Immunoblots of PARP (a nuclear marker) and α-tubulin (a cytosolic marker) serve to confirm the validity of the subcellular fractionation procedure. b, Immunoprecipitation (IP) of the CAD-ICAD complex in normal rat brain cell extracts using anti-ICAD antibody followed by immunoblotting using the anti-CAD/DFF40 antibody. The complex is detected in the nuclear but not in the cytosolic fraction. Normal rabbit IgG (NIgG) and brain protein extracts (Lysate) serve as negative and positive controls, respectively. c, Representative Western blots (left panel) show increases in CAD/DFF40 immunoreactivity in the nuclear fraction after ischemia. Control Western blots show that the purified protein fraction is enriched in the nuclear protein histone but does not contain the cytosolic protein α-tubulin. Shown in the right panel are semiquantitative results of relative abundance of CAD/DFF40 and histone immunoreactivity in the nuclear fraction after ischemia, as determined using densitometric measurement on three individual Western blots performed using three different sets of brain samples. Data are mean ± SEM and represent fold changes in ischemic brains versus sham controls. *p < 0.05 versus sham controls (ANOVA and post hoc Scheffe's tests). d, Immunohistochemical staining of CAD/DFF40 (A1–A4) and caspase-3 (B1–B3) in the hippocampal CA1 sector after ischemia. Compared with that in the control brain (A1) and 24 hr after ischemia (A2), CAD/DFF40 immunofluorescence is markedly increased in the nucleus of CA1 neurons at 72 hr after ischemia (A3). Omission of the primary antibody from immunostaining results in no positive signals (A4). Double-label in the section obtained 72 hr after ischemia shows the colocalization of increased CAD/DFF40 and caspase-3 immunofluorescence in CA1 neurons (B4,blue arrows). Note that caspase-3 immunofluorescence is increased at both 24 hr (B2) and 72 hr (B3) after ischemia, however; only at 72 hr after ischemia does caspase-3 show a nuclear localization (B3). In keeping with delayed cell death in this model, cresyl violet staining demonstrates that CA1 neurons show normal morphology in control brain (C1) and in the brain 24 hr after ischemia (C2) but show pyknotic changes in the brain 72 hr after ischemia (C3, red arrows). As determined using TUNEL staining, DNA fragmentation is not detected in control brain (D1) or at 24 hr after ischemia (D2), but it occurs in a majority of CA1 neurons at 72 hr after ischemia (D3). Note that TUNEL-positive neurons show a condensed, shrunken, or fragmented nucleus (D3, red arrows).

Fig. 5.

Activation of endogenous CAD/DFF40 after cerebral ischemia. a, Representative Western blots show the time course of proteolytic activation of caspase-3 and ICAD cleavage in the cytosolic or nuclear fraction in the hippocampus after ischemia. Note that cleavage product of ICAD (16.5 kDa) and active caspase-3 (17 kDa) was not present in the nucleus until 72 hr after ischemia. Protein extracts from STS-treated (1 μm) cortical neuron cultures serve as the positive control (P) for ICAD cleavage and caspase-3 activation. b, Semiquantitative results of relative abundance of cleavage product of ICAD (16.5 kDa) and active caspase-3 (17 kDa) in the nuclear or cytosolic fraction in the hippocampus after ischemia, as determined using densitometric measurement on three individual Western blots performed using three different sets of brain samples. Data are mean ± SEM and represent fold changes in ischemic brains versus sham controls. *p < 0.05 versus sham controls (ANOVA andpost hoc Scheffe's tests). c, Effects of intracerebral ventricular infusion of z-DEVD-fmk (4.5 μg) on proteolytic activation of caspase-3 and degradation of nuclear ICAD in the hippocampus after ischemia. Note thatz-DEVD-fmk prevents nuclear translocation of active caspase-3 and cleavage of nuclear ICAD 72 hr after ischemia. Immunoblotting of histone serves as the sample-loading control for nuclear protein extracts. d, Effects of intraventricular infusion of z-DEVD-fmk (4.5 μg) on nuclear translocation of CAD/DFF40 in the hippocampus after ischemia. Note thatz-DEVD-fmk does not prevent the increases in the levels of CAD/DFF40 in the nucleus 24 or 72 hr after ischemia.e, Detection of DNA fragmentation-inducing activity in nuclear extracts from the hippocampal CA1 after ischemia. Protein extracts were incubated with genomic DNA from normal brain cells under the experimental conditions described in Materials and Methods, and the resulting DNA fragmentation was detected using terminal deoxynucleotidyl transferase-mediated α-³²P-dideoxyATP labeling and autoradiography. Lanes 1–4, Protein extracts were obtained from sham-operated brain (S) or from brains at 8, 24, or 72 hr after ischemia. Note that nuclear extracts from the 72 hr postischemic brains contain DNA fragmentation-inducing activity (lane 4). Lanes 5–10, The DNA fragmentation-inducing activity in the hippocampal nuclear extracts were inhibited by co-incubation of the mutant ICAD recombinant protein (ICAD_dm) at 0.2 μg/ml (lane 7) or 1 μg/ml (lane 8), by immunodepletion of CAD/DFF40 in the nuclear protein extracts (lane 9), or by intraventricular infusion ofz-DEVD-fmk (lane 10), but not by the endonuclease inhibitor ATA at 0.3 mm (lane 5) or 1 mm (lane 6). The Western blot (right panel) shows the caspase-3-resistant mutant ICAD protein (m) that was used in the DNA fragmentation assays. Compared with the mutant ICAD, the wild-type ICAD protein (w) could be cleaved by caspase-3, generating the 16.5 kDa fragments.f, Detection of endogenous DNA fragmentation in the hippocampus after ischemia. DNA was extracted from the hippocampal CA1 at 8, 24, or 72 hr after ischemia or sham operation. Note that internucleosomal DNA fragmentation is induced at 72 hr but not at 8 or 24 hr after ischemia or sham operation, which is consistent with the time course of increased DNA fragmentation-inducing activity after ischemia.

Fig. 6.

Activation of endogenous CAD/DFF40 in primary cortical cultures. a, Oxygen and glucose deprivation (OGD) induced apoptotic nuclear changes in neurons in the presence of low concentration of MK801 (1 nm). Nuclear morphology was evaluated using propidium iodine (red) and counterstaining with Hoechst 33258 (blue) at 24 hr after 90 min of OGD. A, Nuclei of normal neurons;B, nuclei of OGD-treated neurons in the absence of MK801; C, nuclei of OGD-treated neurons in the presence of MK801. Arrowheads point to nuclei that show characteristic changes of apoptosis. b, Quantitative results show that, in the presence of MK801, OGD significantly increased apoptosis in neuronal cultures. Apoptosis was quantified 24 hr after OGD by counting nuclei that showed chromatin condensation and fragmentation after propidium iodine DNA-staining. Data are mean ± SEM, and each data point represents cell counts of at least 3000 neurons from two independent experiments. **p < 0.01 versus sham controls (ANOVA and post hoc Scheffe's tests). c, DNA ladder. Lane 1, Normal neurons; lane 2, 24 hr after 90 min of OGD, without MK801; lanes 3–4, 24 hr after OGD, in the presence of 1 and 10 nm MK801, respectively; lane 5, 24 hr after OGD, in the presence of both MK801 (1 nm) andz-DEVD-fmk (100 μm). Note that, in the presence of MK801, OGD induces caspase-dependent internucleosomal DNA fragmentation. d, Western blots show the time course of nuclear translocation of active caspase-3 (17 kDa) and proteolytic cleavage of ICAD and PARP (another marker of caspase-3 activation) in the nucleus after 90 min of OGD (in the presence of 1 nm MK801). Nuclear protein was extracted from neurons at 0, 4, 12, or 24 hr after OGD. Cell lysates from STS-treated (1 μm) neurons serve as the positive control (P). e, Detection of DNA fragmentation-inducing activity in nuclear extracts from neurons at 12 or 24 hr (lanes 2–3) after 90 min of OGD (with the addition of 1 nm MK801). Protein extracts were incubated with genomic DNA from normal neurons, and the resulting DNA fragmentation was detected using terminal deoxynucleotidyl transferase-mediated α-³²P-dideoxyATP labeling and autoradiography. This induced DNase activity was inhibited by the addition of mutant ICAD (ICAD_dm) recombinant protein (1 μg/ml) to the reaction mixture (lanes 5–6).f, Effect of induced CAD/DFF40 activity on nuclear morphology. Nuclear extracts from normal neurons (A) or neurons at 12 hr (B) or 24 hr (C) after OGD were incubated with nuclei isolated from normal neurons under conditions described in Materials and Methods, and nuclear morphology was evaluated by propidium iodine staining. Nuclear extracts from OGD-treated neurons result in chromatin fragmentation in isolated nuclei; this activity was inhibited by the addition of ICAD_dm (1 μg/ml) to the reaction mixture (D).