Caspase Activity Is Involved in, but Is Dispensable for, Early Motoneuron Death in the Chick Embryo Cervical Spinal Cord

MCN

Molecular and Cellular Neuroscience 18, 168 –182 (2001) doi:10.1006/mcne.2001.1009, available online at http://www.idealibrary.com on

Caspase Activity Is Involved in, but Is Dispensable for, Early Motoneuron Death in the Chick Embryo Cervical Spinal Cord Hiroyuki Yaginuma,* ,1 Nobuko Shiraiwa, †,‡ Takako Shimada,* Keiji Nishiyama,* Jason Hong, † Siwei Wang, † Takashi Momoi, § Yasuo Uchiyama, ¶ and Ronald W. Oppenheim † *Department of Anatomy, School of Medicine, Fukushima Medical University, Fukushima 960-1295, Japan; †Department of Neurobiology and Anatomy and the Neuroscience Program, Wake Forest University School of Medicine, Winston–Salem, North Carolina 27157; § Division of Development and Differentiation, National Institute of Neuroscience, NCNP, Kodaira, Tokyo 187-8502, Japan; ¶Department of Cell Biology and Anatomy I, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan; and ‡ Department of Neurology, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8575, Japan

We examined the role of caspases in the early programmed cell death (PCD) of motoneurons (MNs) in the chick embryo cervical cord between embryonic day (E) 4 and E5. An increase in caspase-3-like activity in MNs was observed at E4.5. Treatment with an inhibitor of caspase3-like activity, Ac-DEVD-CHO, for 12 h blocked this increase and revealed that caspase-3-like activity is mainly responsible for DNA fragmentation and the nuclear changes during PCD but not for degenerative changes in the cytoplasm. When a more broad-spectrum caspase inhibitor was used (bocaspartyl (OMe)-fluoromethyl ketone, BAF), the appearance of degenerative changes in the cytoplasm was delayed by at least 12 h. However, following treatment with either Ac-DEVD-CHO or BAF for 24 h, the number of surviving healthy MNs did not differ from controls, indicating a normal occurrence of PCD despite the inhibition of caspases. These results suggest that caspase cascades that occur upstream of and are independent of the activation of caspase-3-like activity are responsible for the degenerative changes in the cytoplasm of dying cervical MNs. These data also suggest that, although one function of caspases may be to facilitate the kinetics of PCD, caspases are nonetheless dispensable for at least some forms of normal neuronal PCD in vivo.

1 To whom correspondence and reprint requests should be addressed. Fax: ⫹81-24-549-8811. E-mail: [email protected].

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INTRODUCTION Programmed cell death (PCD) is a fundamental component of normal development and adult homeostasis in virtually all multicellular organisms (for review, see Glu¨cksmann, 1951; Saunders, 1966; Wyllie et al., 1980; Raff, 1992; Schwartz and Osborne, 1993). In the chick embryo spinal cord, two types of PCD of motoneurons (MNs) have been observed: one occurs at a relatively late stage and the other at an early stage of MN differentiation. The first type of PCD occurs mainly between embryonic day (E) 6 and E10 in MN populations at all levels of the spinal cord, including the cervical region. Approximately 50% of MNs die during this period (Hamburger, 1958; Hollyday and Hamburger, 1976; Chu-Wang and Oppenheim, 1978; Hamburger and Oppenheim, 1982). The survival of MNs at this stage is dependent on their interaction with muscle targets (Hamburger, 1958; Hollyday and Hamburger, 1976; Hamburger and Oppenheim, 1982; Oppenheim, 1991; Caldero et al., 1998). In the second type of MN degeneration, PCD occurs only in the nonlimb innervating cervical spinal cord between E4 and E5 (Levi-Montalcini, 1950, 1964; Yaginuma et al., 1996). A similar early cervical MN death may also occur in mammals (Yamamoto and Henderson, 1999). In the chick these MNs cannot be rescued by increasing the size of peripheral targets or by treatment with neuromuscular 1044-7431/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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blocking agents, muscle extracts, or most neurotrophic agents (Yaginuma et al., 1996), manipulations that, by contrast, rescue MNs from the first (late) type of cell death. Caspase-3 is the most widely investigated member of the caspase family and is involved in the execution of cell death in vertebrates. It is known that caspase-3 activity is required at the step where a protease cascade pathway converges. Activated caspase-3 cleaves ICAD/DFF45 (Liu et al., 1997; Sakahira et al., 1998), leading to activation of CAD/DFF40 that causes DNA fragmentation in dying cells (Enari et al., 1998; Liu et al., 1998; Sakahira et al., 1998). Many previous in vitro studies have shown that various kinds of stimuli that normally induce apoptotic cell death characterized by chromatin condensation and DNA fragmentation failed to do so in cells that were caspase-3 deficient or in which caspase-3 or other caspases were inhibited (Ja¨nicke et al., 1998; Woo et al., 1998; Zheng et al., 1998; Bortner and Cidlowski, 1999; Cregan et al., 1999; Ferrer, 1999; Stefanis et al., 1999; Tanabe et al., 1999; Xue et al., 1999; D’Mello et al., 2000). Studies of caspase-3- and caspase-9-deficient mice have demonstrated that these mice exhibit reduced PCD and lack DNA fragmentation in the early embryonic central nervous system, resulting in abnormal organization (exencephaly) of the cerebral cortex (Kuida et al., 1996, 1998; Hakem et al., 1998; Kuan et al., 2000). However, it has also been suggested that normal amounts of PCD are occurring in the brain stem and spinal cord of these mutants (Kuida et al., 1996, 1998; Hakem et al., 1998) and a recent study has confirmed this (Oppenheim et al., 2001). The PCD of some nonneuronal cells also appears normal (but delayed) in these mutants (Cecconi et al., 1998; Hakem et al., 1998; Kuida et al., 1998; Yoshida et al., 1998; Chautan et al., 1999). These studies indicate that the involvement of caspase-3 and other caspases in PCD may differ depending on cellular phenotype, the stage at which PCD occurs, and the specific stimuli that trigger death. In a previous study, we reported that inhibitors of the caspase-1-like protease (ICE) fail to rescue the early dying MNs in the cervical cord of the chick embryo (Milligan et al., 1995). These results suggested that there may be other signals and pathways that mediate this form of PCD. In the present study, we have investigated the involvement of caspase-3-like activity as well as other upstream caspases in the MN death that occurs in the cervical spinal cord between E4 and E5. We observed that caspase-3-like activity is expressed in these MNs during PCD in vivo and that inhibitors of caspase3-like activity can arrest the nuclear changes but not the cytoplasmic events that occur during this type of PCD.

Furthermore, we found that although other upstream caspases are involved in the more extensive nuclear and cytoplasmic changes, inhibition of these caspases by the pan-caspase inhibitor bocaspartyl (OMe)-fluoromethyl ketone (BAF) delays but does not prevent this type of PCD.

RESULTS In a previous study, we determined (by counting the number of pyknotic cells) that early MN death in the cervical cord of the chick embryo begins at stage (st) 23 (E4), reaches a peak at st 24 (E4.5), and ends by st 27 (E5.5) (Yaginuma et al., 1996). Although numerous studies have demonstrated that there are many intracellular processes or steps that precede the morphological manifestation of PCD, in describing our results, we will use the term “onset” or “initiation” of PCD to mean the time of first observable morphological degenerative changes in MNs and the “end” of PCD as the disappearance of pyknotic cells (i.e., the stage/age when pyknotic degenerating cells are no longer present). Analysis of Enzyme Activity Caspase-3-like activity in the embryonic spinal cord was measured before, during, and after the period of cervical MN death using Ac-DEVD-MCA as a substrate. Caspase-3-like activity was increased in the cervical segments at st 24 and this activity was almost completely inhibited by Ac-DEVD-CHO (Fig. 1). When activity was measured after the cervical segments were subdivided into ventral and dorsal halves, caspase-3like activity was found to be localized to the ventral, MN-containing region. Examination of Activated Caspase-3-like Immunoreactivity To more directly assess the involvement of caspase3-like activity in early PCD in the cervical segments, the pattern of expression of activated caspase-3 in the cervical segments was examined using a cleavage sitedirected antibody against caspase-3 (anti-p20/17). This antibody specifically recognizes activated caspase-3 in mouse, human (Kouroku et al., 1998; Urase et al., 1998; Isahara et al., 1999), and chicken (T. Momoi, unpublished observation) tissues. Double labeling with TUNEL staining revealed that activated caspase-3-like immunoreactivity was expressed in dying MNs in the ventral horn from st 23⫹ to st 26 (Fig. 2). However,

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FIG. 1. Caspase-3-like activity in the spinal cord of chick embryos from st 22 to st 26 (E3.75–E5). Enzymatic activities are indicated as unit activities per milligram of protein. Mean activities were obtained from at least three independent experiments and at least five spinal cords were used for each measurement. C, cervical cord; Th, thoracic cord. *P ⬍ 0.01, **P ⬍ 0.001, t test.

immunoreactivity was also observed in cells whose nuclei were TUNEL-negative and in which the cellular morphology was apparently normal (Fig. 2, arrow). Activated caspase-3-like immunoreactivity was never observed in regions of the spinal cord where PCD does not occur (data not shown) nor was caspase-3-like immunoreactivity ever seen in cells in advanced stages of apoptosis (i.e., at a stage when apoptotic bodies occur).

In Ovo Treatment with Caspase Inhibitors To elucidate the role of caspases in vivo, we examined the effects of treatment with caspase inhibitors. We used Ac-DEVD-CHO, an inhibitor that is relatively specific for caspase-3, caspase-6, and caspase-7, and BAF, an inhibitor that acts on most if not all caspases. Treatment was began several hours before the onset of PCD (st 23⫺) and embryos were fixed either 12 h (st 24) or 24 h later (st 25⫹/26⫺). The detailed morphology of dying cells was examined and the numbers of pyknotic cells and TUNEL-positive cells were counted (Figs. 3– 8). Treatment with Ac-DEVD-CHO for 12 h. To assess caspase-3-like activity following Ac-DEVD-CHO treat-

ment, we performed immunohistochemistry using the antibody against caspase-3 and measured cleavage of AcDEVD-MCA. Immunolabeling for activated caspase-3 was barely detectable (data not shown) and the enzymatic activity was reduced to 26% of the control (control:treated ratio ⫽ 0.0038U:0.001U). This indicates that Ac-DEVDCHO at the doses used here not only effectively inhibits caspase-3-like activity for up to 12 h in vivo but also prevents the processing of inactive procaspase-3 to the active form. In sections stained with hematoxylin and eosin (HE), the morphology of dying cells at st 24 treated with 400 ␮g of Ac-DEVD-CHO was distinctly different from controls (Figs. 3A and 3B). In controls, the nuclei of dying cells were round in shape, and the chromatins were condensed and darkly stained by hematoxylin (nuclear pyknosis) and subsequently became fragmented (Fig. 3A). By contrast, the nuclei of cells from Ac-DEVDCHO-treated embryos exhibited only moderate chromatin condensation and pyknosis and had a crenulated shape (Fig. 3B). When we counted the number of dying (pyknotic) cells in HE-stained sections, including the atypical, moderately pyknotic cells, we found that there was no significant difference in the number of pyknotic

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FIG. 2. A photomicrograph of the triple staining of the active form of caspase-3 (green), TUNEL (red), and DAPI (blue) in the ventral horn region of the E4.5 chick cervical spinal cord. Arrowheads indicate the active forms of caspase-3 in cells whose nuclei had become TUNEL-positive. The arrow indicates a cell in which activated caspase-3 is expressed but whose nucleus is not TUNEL labeled and which lacks chromatin aggregation by DAPI staining which is therefore apparently intact. Scale bar is 10 ␮m.

cells between controls and Ac-DEVD-CHO-treated embryos (Fig. 4A). To exclude the possibility that the initiation of the Ac-DEVD-CHO treatment was too late to inhibit the process of nuclear pyknosis, in two groups treatment was started at st 21⫹ (8 h before the initiation of morphological manifestation of PCD). However, again the number of moderately pyknotic cells was not reduced (data not shown). To exclude the possibility that the doses used were not effective in inhibiting pyknosis, we have also used doses higher than 400 ␮g. However, even after treatment with 1.2 mg of AcDEVD-CHO, the number of moderately pyknotic cells was not reduced (Fig. 4A). By contrast, examination of DNA fragmentation using the TUNEL method revealed that the number of cells with DNA fragmentation was significantly reduced following treatment with AcDEVD-CHO (Figs. 3E–3H) and this effect was dosedependent (Fig. 4B). To determine whether the cells that are moderately pyknotic and TUNEL-negative in the Ac-DEVD-CHO-treated embryos retain a MN phenotype, we examined the expression of a cell (MN)-spe-

FIG. 3. Photomicrographs showing the morphology, DNA fragmentation, expression of a MN-specific marker (Islet), and expression of the apoptosis-specific protein (ASP) by MNs in the cervical cord at st 24 following treatment with Ac-DEVD-CHO and BAF for 12 h. Scale bar in A is 10 ␮m for A–F. Scale bar in G is 10 ␮m for G–L. (A–C) Semithin sections (2-␮m thick) stained with hematoxylin and eosin. (D–F) TUNEL reaction. (G–I) Cryostat sections immunolabeled for Islet-1 (red) and DAPI (blue) showing the expression of this MN-specific marker. (J–L) Triple staining of ASP (green), Islet-1 (red), and DAPI (blue). (A, D, G, and J) Controls. (B, E, H, and K) Treated with 400 ␮g of Ac-DEVD-CHO. (C, F, I, and L) Treated with 200 ␮g of BAF. Arrows in A and B indicate examples of pyknotic neurons. The insets in A–C show higher magnifications of the morphologies of typical pyknotic neurons (A), modified degenerating neurons (B), and darkly stained (presumptive degenerating) neurons (C). DNA fragmentation was inhibited in the embryos treated with Ac-DEVD-CHO (E) and BAF (F). Arrows in H indicate cells with smaller and irregular-shaped nuclei that are Islet-1 negative in the ventral horn. The insets in J–L show higher magnifications of the cells that express both Islet-1 and ASP. Note that, following BAF treatment, most cells in the ventral horn retain Islet-1 (I) and that ASP-positive cells are localized to the lateral portion of the ventral horn (L).

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FIG. 4. Quantitative analyses of the effects of caspase inhibitors. (A, B) The numbers of pyknotic cells (A) and TUNEL-positive cells (B) per section (mean ⫾ SD) in the C10 segment of chick embryo spinal cord treated with caspase inhibitors for 12 h. Pyknotic profiles and TUNEL-positive cells were counted in every 6th section as described previously (Yaginuma et al., 1996). (C) The number of Islet-1-positive cells per section (mean ⫾ SD) following 12 h of treatment with caspase inhibitors. The number of Islet-1positive nuclei was counted in every 10th section of the C8 to C11 segments.

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cific marker, Islet-1. In a previous study, we reported that Islet-1 is degraded and disappears during the process of cervical PCD and its disappearance follows the initiation of DNA fragmentation (Yaginuma et al., 1996). Most of the surviving healthy looking cells in the ventral horn region of the cervical cord in control embryos expressed Islet-1, whereas Islet-1 expression was no longer detectable in dead or dying MNs (Fig. 3G). Following Ac-DEVD-CHO treatment, many Islet-1-negative cells with irregular-shaped nuclei were observed in the ventral horn region (Fig. 3H). Because at st 23, when PCD begins in this population, virtually all neurons in the ventral horn region express Islet-1 (Yaginuma et al., 1996), it is likely that these Islet-1-negative cells have lost the expression of this MN-specific marker despite Ac-DEVD-CHO treatment. This suggests that AcDEVD-CHO treatment does not arrest this or perhaps other degenerative changes occurring in these cells and that the disappearance of Islet-1 during cell death is independent of caspase-3-like activity. We also examined the expression of an apoptosis-specific marker, apoptosis-specific protein (ASP), which is recognized by anti-c-Jun/AP-1 antibodies owing to cross-reaction (Terwel and van de Berg, 2000). In the ventral horn of control embryos, ASP was strongly expressed by dead cells that had already lost Islet-1 expression as well as by cells that still expressed Islet-1 (Fig. 3J). Following Ac-DEVD-CHO treatment, expression of ASP appeared weaker than that in controls, but nonetheless ASP expression was observed in both Islet-1-positive cells and Islet-1-negative cells with irregular-shaped nuclei (Fig. 3K). We have also examined the detailed morphology of pyknotic cells using electron microscopy. In control embryos at st 24, dying cervical MNs exhibited a typical apoptotic morphology with condensation of the cytoplasm (Fig. 5A). The cell nuclei were small and amorphous in appearance due to condensed chromatin and subsequently became fragmented; aggregated ribosomes and a few clear vacuoles were often observed in the cytoplasm. In comparison, following treatment with Ac-DEVD-CHO, many aberrantly degenerating cells were observed. The soma/cytoplasm exhibited degenerative changes characterized by shrinkage of cell size, condensation, increased electron density, and accumulation of clear vacuoles and aggregated ribosomes (Figs. 5B and 5C). By contrast, the nuclei of these same cells appeared relatively normal; although most nuclei exhibited a slight increase in electron density and an irregular shape as well as a slight aggregation of chromatin, the extreme shrinkage and fragmentation of nuclei observed in degenerating control cells were rarely

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FIG. 5. Electron micrographs showing the ultrastructural morphology of degenerating cells in the ventral horn of the cervical spinal cord of the chick embryo following treatment with Ac-DEVD-CHO and BAF for 12 h. (A) Typical example of the type of degeneration found in a st 24 normal embryo. Condensation of chromatin (n), aggregated ribosomes (arrows), and increased electron density of the cytoplasm are distinct. (B–D) St 24 embryos following 12 h of treatment with Ac-DEVD-CHO. n, nucleus. Note that a degenerating neuron is engulfed by a macrophage-like cell (m) in D (arrows). (E) A st 24 embryo following 12 h of treatment with BAF. Note the many small cells with small nuclei and slightly electron-dense cytoplasm. (F) Higher magnification of E. n⬘, a nucleus of an apparently healthy cell. n, the nucleus of a small “degenerating” cell. Scale bars are 2 ␮m.

174 observed (Figs. 5B and 5C). We noted that these aberrantly degenerating cells were often already engulfed by processes of presumptive macrophages (Fig. 5D), indicating that the cells were actually dead or dying. Treatment with Boc-D-FMK (BAF) for 12 h. To investigate the involvement of other caspases that may not have been inhibited by Ac-DEVD-CHO, we used the pan-caspase inhibitor BAF. After treatment with 200 ␮g of BAF for 12 h, the number of pyknotic cells in HE-stained preparations was less than that in controls. Even the moderately pyknotic cells that were often observed after treatment with Ac-DEVD-CHO were rarely seen. Instead, there were many abnormal cells that were smaller and eosinophilic (i.e., stained darker, Fig. 3C) compared to other, presumptive healthy cells. These abnormal cells were mainly located in the lateral portion of the ventral horn and they lacked DNA fragmentation (Figs. 3F and 4B) and immunoreactivity for the active form of caspase-3 (data not shown), and also retained Islet-1 expression (Fig. 3I). By using their unusual morphology (dark HE staining and reduced nuclear size) as a criterion for identification (see Experimental Methods), we counted the number of these cells on E4.5 and found that they were similar to the number of pyknotic MNs in control embryos and to the number of moderately pyknotic MNs in the Ac-DEVD-CHO treated embryos (25.3 ⫾ 6.1 per section, mean ⫾ SD, n ⫽ 4; compare with Fig. 4). These data suggest that these are, in fact, presumptive degenerating MNs in which the typical morphological changes of apoptosis have been delayed. Further support for this is the fact that the total number of Islet-1-positive MNs in these BAF-treated embryos is increased significantly (Fig. 4C) and the observation that many of these cells exhibit both Islet-1 and ASP immunoreactivity (Fig. 3). The number of cells that expressed both Islet-1 and ASP was assessed using confocal laser microscopy and was 29.5 ⫾ 3.1 per section (mean ⫾ SD, n ⫽ 4). This corresponds to approximately 26% of the total population of Islet-1-positive cells in the ventral horn at this stage. Electron microscopic observations confirmed that there were many smaller cells with irregular-shaped small nuclei and slightly electron-dense cytoplasm but with apparently normal organelles and nuclear chromatin, and these tended to occur in clusters (Figs. 5E and 5F). Together, the aberrant morphology, ASP immunolabeling, and cell counts indicate that these are likely presumptive degenerating MNs. Results similar to those described above were found when BAF and Ac-DEVDCHO were given together (data not shown). Treatment with Ac-DEVD-CHO and BAF for 24 h. To assess the effects of more prolonged caspase inhibi-

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FIG. 6. Photomicrographs showing the ventral horn region of the cervical cord of st 25⫹/26⫺ control embryos (A, D, G) and embryos treated with Ac-DEVD-CHO (B, E, H) and BAF (C, F, I) for 24 h. (A–C) Hematoxylin and eosin staining. (D–F) TUNEL reaction. (G–I) Triple staining of apoptosis-specific protein (ASP, green), Islet-1 (red), and DAPI (blue). Arrows in A–C indicate examples of pyknotic neurons. The insets in A–C show higher magnifications of pyknotic cells. Arrowheads in B and E indicate clusters of cell debris engulfed by macrophage-like cells. Note that most of the debris does not show nuclear pyknosis but is positively stained by TUNEL reaction (compare B and E) and by immunohistochemistry for ASP (arrowheads in H). Many cells that are Islet-1-negative and only weakly express ASP were localized in the most lateral region of the ventral horn in the BAF-treated embryos (arrowheads in I). Scale bar in A is 10 ␮m for A–F. Scale bar in G is 10 ␮m for G–I.

tion, we examined embryos treated for 24 h with either Ac-DEVD-CHO or BAF. This duration of treatment did not appear to affect the general development of embryos. In control E5 embryos, the number of pyknotic cells, TUNEL-positive cells, and ASP-positive cells was reduced compared to those in E4.5 (Figs. 6A, 6D, 6G, and 7A). In embryos treated with Ac-DEVD-CHO, the number of pyknotic cells was slightly reduced from the number present at 12 h but was still significantly higher than that of control embryos (Figs. 6B and 7A) and the number of TUNEL-positive cells was increased compared to that at 12 h (Figs. 7B, compare Fig. 6E with Fig.

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FIG. 7. (A, B) Line graphs showing changes in the numbers of pyknotic cells (A) and TUNEL-positive cells per section (mean ⫾ SD) in the C10 segment at 12 and 24 h of treatment with caspase inhibitors. (C) Bar graph showing the number of Islet-1-positive cells per section (mean ⫾ SD) following 24 h of treatment with caspase inhibitors. The number of Islet-1-positive nuclei was counted in every 10th section of the C8 to C11 segments.

3E). We very often observed clusters of debris from dead cells being engulfed by macrophages (Figs. 6B and 6E). ASP-like immunoreactivity was observed in asso-

ciation with irregular-shaped cell nuclei that did not express Islet-1 and with cell debris that had already been engulfed by macrophages (Fig. 6H). Electron microscopic observations revealed that some cells exhibited distinct morphological signs of apoptosis, whereas others only exhibited the aberrant degenerative changes observed at 12 h (Fig. 8A). Macrophages were often observed engulfing dead cells (Fig. 8B). Following treatment with BAF for 24 h, the number of pyknotic cells increased to a level comparable to that in embryos treated with Ac-DEVD-CHO. However, the number of TUNEL-positive cells was significantly less than that in either controls or Ac-DEVD-CHO-treated embryos (Fig. 7B). We observed many moderately pyknotic cells by light microscopy (Fig. 6C) and these appeared to correspond to the aberrant degenerating cells observed in the electron microscope (Fig. 5C). The morphology of some of these aberrantly degenerating cells was very similar to that observed following 12 h treatment with Ac-DEVD-CHO (Fig. 8C). However, we also noted in many of the degenerating cells following BAF treatment for 24 h, that autophagic vacuoles were conspicuous (Fig. 8D). Triple labeling by Islet-1, ASP, and DAPI revealed that there were many irregularshaped and small nuclei that were Islet-1 negative but that weakly expressed ASP-like immunoreactivity and were located in the most lateral portion of the ventral horn (Fig. 6I). The total number of surviving MNs which expressed Islet-1 was unchanged following 24 h treatment with Ac-DEVD-CHO or BAF compared with control embryos (Fig. 7C). Similar results were obtained in embryos treated with both BAF and Ac-DEVD-CHO for 24 h (data not shown). To exclude the possibility that the apparent lack of a rescue effect by BAF was owing to our treatment schedule (6-h intervals), we performed one experiment in which BAF was administered every 3 h so that a total of 1.5 times more BAF was given. However, the total number of surviving MNs which express Islet-1 was unchanged (data not shown). From these results we conclude that despite the often unusual morphology of degenerating MNs following caspase inhibition, the extent of PCD and cell loss is comparable to controls.

DISCUSSION In the present study, we have shown that caspase-3like activity is involved in the normal MN death that occurs in the cervical cord of the early chick embryo. Caspase-3-like activity is mainly responsible for DNA

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FIG. 8. Electron micrographs showing the ultrastructural morphology of degenerating cells in the ventral horn of the cervical spinal cord of chick embryos following treatment with Ac-DEVD-CHO (A, B) and BAF (C, D) for 24 h. Note that the debris from dead cells is contained in a macrophage-like cell (B). Arrowheads in the inset of D show typical autophagic vacuoles. Scale bars are 2 ␮m.

fragmentation and the nuclear changes during PCD. Although the inhibition of caspase-3-like activity delayed the rate of PCD, degeneration nonetheless occurred. Furthermore, when a broad-spectrum caspase inhibitor was used (BAF), the delay in PCD was increased, although eventually normal numbers of MNs underwent PCD. Caspase activity was measured using synthetic peptide substrates. Although caspase-3 cleaves Ac-DEVDMCA, other caspases such as caspase-7 and -8 are also known to cleave Ac-DEVD-MCA (Thornberry et al., 1997). To demonstrate the specific involvement of caspase-3 in the process of PCD, we utilized an antiserum (anti-p20/17) that reacts specifically with the p20/17 cleaved fragment of caspase-3. The results unequivocally demonstrate that caspase-3 is activated during early cervical MN death and this activation is abolished for up to 12 h following in vivo treatment with Ac-DEVD-CHO and BAF. Although cervical MNs were not labeled by the TUNEL method following treatment with either AcDEVD-CHO or BAF, they were nonetheless dying based on their ultrastructure and on the number of

surviving healthy MNs present at the end of the normal period of PCD on E5. Cells which appear only moderately pyknotic in the light microscope following AcDEVD-CHO treatment exhibited ultrastructural degenerative changes similar to those observed in normal embryos except for the absence of changes in the nuclei. These degenerative changes appear to be required for PCD because it was confirmed that such cells are phagocytosed by macrophages. The fact that the number of surviving healthy MNs following treatment with caspase inhibitors does not differ from controls is also consistent with the occurrence of PCD. It is known that the role of caspase-3 in PCD occurs at a critical downstream site involving the convergence of several proteases (Darmon et al., 1995; Martin et al., 1996; Quan et al., 1996; Li et al., 1997; Nagata, 1997; Kuida et al., 1998). Activated caspase-3 cleaves ICAD/ DFF45 (Liu et al., 1997; Sakahira et al., 1998), leading to activation of CAD/DFF40 that results in DNA fragmentation (Enari et al., 1998; Liu et al., 1998; Sakahira et al., 1998). Caspase-3 also activates caspase-6, which then mediates the shrinkage and fragmentation of nuclei (Hirata et al., 1998; Kawahara et al., 1998). Previous in

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vitro studies have shown that various kinds of stimuli that normally induce apoptosis (including DNA degradation and chromatin condensation) fail to do so in cells that lack caspase-3 (Ja¨nicke et al., 1998; Woo et al., 1998; Zheng et al., 1998; Bortner and Cidlowski, 1999; Cregan et al., 1999; Ferrer, 1999; Stefanis et al., 1999; Tanabe et al., 1999; Xue et al., 1999; D’Mello et al., 2000). Accordingly, caspase-3 appears to be primarily involved in the nuclear changes that occur during PCD. Double-staining of MNs with anti-p20/17 and TUNEL revealed that activated caspase-3-like immunoreactivity was also observed in MNs whose nuclei were TUNEL-negative and in which cellular morphology was apparently normal. One likely explanation of this observation is that these MNs are in early stages of PCD when activation of caspase-3 occurrs but prior to DNA fragmentation and nuclear shrinkage. A similar expression of activated caspase-3 preceding DNA fragmentation has often been observed in cultured PC12 cells following NGF depletion (Y. Uchiyama, unpublished observation). However, because of technical limitations, we cannot entirely exclude the possibility that activated caspase-3 is only transiently expressed in some cells and may not actually induce cell death in this situation. Following Ac-DEVD-CHO treatment, DNA fragmentation was inhibited and most MN nuclei did not exhibit the extreme shrinkage, condensation, or fragmentation normally associated with apoptosis. These morphological features are similar to those observed in dying embryonic MNs from caspase-3-deficient mice (Oppenheim et al., 2001), indicating that the Ac-DEVDCHO treatment used here actually suppresses caspase-3 activity in the chick embryo. Motoneurons in the caspase-3-deleted mice exhibited reduced TUNEL labeling, an absence of activated caspase-3 immunoreactivity, and ultrastructural changes (e.g., cytoplasmic vacuoles, reduced chromatin condensation, dilated cytoplasmic organelles) almost identical to those observed in our Ac-DEVD-CHO-treated chick MNs. Cell counts in these mice show that normal numbers of MNs have undergone PCD (Oppenheim et al., 2001). These results indicate that the same execution machinery for PCD, which has been primarily studied in mammalian cells in vitro, is also utilized by developing avian neuronal cells which undergo normal PCD in vivo. Following treatment with the pan-caspase inhibitor BAF for 12 h, cervical MNs did not exhibit an apoptotic morphology. However, many MNs did exhibit an abnormal morphology and when cells of this type were counted their numbers were comparable to the number of pyknotic cells in control and Ac-DEVD-CHO-treated embryos at E 4.5. The difference in morphology be-

177 tween the effects of Ac-DEVD-CHO and BAF can be explained by differences in their specificity for caspase family members rather than inadequate concentrations of Ac-DEVD-CHO. We were not able to observe effects similar to those of BAF using 1.2 mg of Ac-DEVD-CHO, a dose six times greater than that of BAF. MNs from embryos treated with this high dose of Ac-DEVD-CHO still exhibit the modified pyknotic morphology seen at lower doses. The results following BAF treatment suggest that other caspases not inhibited by Ac-DEVDCHO play key roles in the apoptotic morphological changes in the cytoplasm. We are currently investigating the involvement of specific upstream caspases in these events. Recent studies have suggested that DNA fragmentation itself is not necessary for PCD. Cells which lack caspase-3 activity or that overexpress ICAD undergo PCD without DNA fragmentation (Xiang et al., 1996; McCarthy et al., 1997; Ja¨nicke et al., 1998; Sakahira et al., 1998; Woo et al., 1998; Zheng et al., 1998; Cregan et al., 1999; Stefanis et al., 1999; Oppenheim et al., 2001). Our results are compatible with these studies and, in addition, indicate that neither DNA fragmentation nor the other biological actions of either caspase-3 or of other caspases that are inhibited by Ac-DEVD-CHO or BAF are essential for PCD of cervical MNs in the chick embryo. The effects of prolonged (24 h) treatment with AcDEVD-CHO or BAF suggest that one role of caspases in vivo is to facilitate the removal of cells that are destined to die by accelerating degeneration of cell nuclei, fragmentation of DNA, phagocytosis, and digestion by other cells. Following treatment with caspase inhibitors for 24 h, there were significantly more pyknotic cells in the cervical ventral horn, whereas in these same embryos the number of Islet-1-positive MNs was comparable to controls. This suggests that the time required for PCD of each cell was retarded rather than that more MNs undergo PCD following prolonged (24 h) treatment with caspase inhibitors. Similar results from in vitro studies have been previously reported (McCarthy et al., 1997; Zheng et al., 1998; Cregan et al., 1999; Stefanis et al., 1999). The dying or dead cervical MNs studied by us are rapidly phagocytosed and digested, and by st 26 (E5) virtually all pyknotic cells have disappeared (Yaginuma et al., 1996). Although further studies are needed to identify which step of this process is affected by caspase inhibitors and whether phagocytes are involved when caspases are inhibited, our findings provide the first in vivo evidence that the significance of caspase activity in at least some forms of neuronal PCD is normally to facilitate the processing of dying cells

178 (McCarthy et al., 1997; Sakahira et al., 1998) but without being required for PCD itself. It is known that caspase-3- and caspase-9-deficient mice have reduced PCD in the early embryonic brain, resulting in abnormal organization (exencephaly) of the cerebral cortex (Kuida et al., 1996, 1998; Hakem et al., 1998). However, the structure of the spinal cord and brainstem appears normal in these mice (Kuida et al., 1996, 1998; Hakem et al., 1998). A recent study has revealed that following the period of cell death there are normal numbers of MNs, DRG cells, spinal interneurons, and sympathetic neurons in postnatal and embryonic caspase-3- and caspase-9-deficient mice and that during the cell death period there are normal numbers of degenerating MNs, but decreased numbers of TUNEL-positive cells (Oppenheim et al., 2001). Collectively, these data from chick and mouse embryos indicate that the involvement of caspases in PCD differs depending on the type of neuron, the stage at which PCD occurs, and the specific stimuli that trigger death. It has been reported that during the normal death of chicken lumbar MNs caspase-3-like activity is involved and that peptide inhibitors of caspase-3 can reduce the number of pyknotic MNs in vivo and prevent the PCD of MNs following trophic factor deprivation in vitro (Li et al., 1998). However, peptide inhibitors of caspase-3 do not rescue these same MNs from cell death following early limb-bud removal (Caldero et al., 1998). The role of caspase-3-like activity in the normal PCD of MNs in the chick embryo needs to be reexamined in light of this apparent discrepancy and in view of the present results indicating that in the absence of caspase activity PCD is delayed but not prevented. Taken together, these studies suggest that, for at least some forms of normal PCD, activation of caspase-3 is not an essential process and that other death pathways acting parallel to caspase-3 activation can ultimately kill the cell. In the absence of detailed measures of caspase inhibition following BAF treatment, we cannot entirely exclude the possibility that some downstream caspase activity may remain and mediate the aberrant, delayed PCD we observe. However, if we assume, as seems likely, that the doses of BAF used by us can, in fact, effectively inhibit all of the caspase-mediated execution machinery, then what mechanisms are mediating PCD in this situation? One possible candidate is noncaspase-mediated autophagy. Many studies have demonstrated that autophagy occurs in various kinds of normal cell death and following treatment with celldeath-inducing stimuli (Clarke, 1990; Nitatori et al., 1995; Jia et al., 1997; Ohsawa et al., 1998; Isahara et al., 1999; Xue et al., 1999). For example, when caspase ac-

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tivity is inhibited, autophagic mechanisms and the activity of cathepsin D, a lysosomal enzyme, can mediate PCD of sympathetic neurons and PC12 cells following NGF or serum deprivation in vitro (Isahara et al., 1999; Xue et al., 1999). Autophagy may also be involved in cervical MN death following caspase inhibition. The appearance of apparent autophagic vacuoles in dying MNs following caspase inhibitor treatment supports this idea. Although many of these vacuoles appear devoid of lysosomal material, some of them, especially those observed in the degenerating cells following BAF treatment for 24 h, appear to be autophagic vacuoles, defined as vacuoles that are limited by double membranes and that contain cytoplasmic organelles (Dunn, 1990). Therefore, although it is possible that autophagy is occurring in dying chick MNs following caspase inhibition, more studies are needed to confirm this. Interestingly, dying MNs in caspase-3-deficient mice exhibit similar cytoplasmic vacuoles (Oppenheim et al., 2001). The presence of ASP immunoreactivity in normal PCD (Ayala et al., 1999) suggests that even in the absence of caspase inhibition, autophagy may play a contributory role in the developmental degeneration of neurons. One interesting observation from our results is that, following BAF treatment, degenerating MNs that are ASP and Islet-1 positive, but later become Islet-1 negative, are localized in the lateral portion of the ventral horn, whereas, by contrast, in controls and Ac-DEVDCHO-treated embryos, degenerating MNs are distributed throughout the ventral horn. One possible explanation for this localized distribution is that in the BAF-treated embryos, MNs that normally initiate degeneration between E4 and E4.5 (st 24) are delayed in PCD and survive until E4.5 when they have completed their migration. In a previous study, we reported that the majority of degenerating MNs at E4.5 became postmitotic between E3 and E3.5 (Yaginuma et al., 1996). Motoneurons that are born at that stage are initially located in the medial region of the ventral horn on E4 and then migrate to more lateral regions between E4 and E4.5 (N. Kobayashi and H. Yaginuma, unpublished observation). Therefore, it is likely that many of these MNs are still migrating laterally when they first initiate degeneration. Normally, degeneration progresses rapidly and the degenerating MNs die before they reach their normal lateral destination. BAF may retard the kinetics of degeneration such that these MNs now remain alive and motile so as to reach the lateral portion of the ventral horn before completing PCD. It is well known that functionally related MNs are segregated together, forming MN pools in the medial motor column of the cervical and thoracic segments (Gutman et

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al., 1993) as well as in the lateral motor column of the brachial and lumbosacral segments (Hollyday et al., 1977; Landmesser, 1978; Hollyday and Jacobson, 1990). Therefore, the fact that ASP-positive MNs were localized in the lateral region of the ventral horn following BAF treatment suggests that these MNs belong to a functionally related subpopulation with similar birthdates. Although further investigations are needed, the present results, together with our previous observations (Yaginuma et al., 1996), suggest that the PCD of MNs in the cervical cord of avian embryos may involve specific subpopulations of MNs.

EXPERIMENTAL METHODS Embryos Fertilized chicken eggs were obtained from Daiichi Farm (Akagi, Gunma, Japan). Eggs were incubated in the laboratory (37.6°C, 60% humidity) until the embryos reached the desired stages. Substrates and Inhibitors of Caspases Ac-DEVD-MCA and Ac-DEVD-CHO were obtained from Peptide Institute (Minoo, Osaka, Japan). Bocaspartyl (OMe)-fluoromethyl ketone (BAF) was purchased from Enzyme System Products (Livermore, CA). Measurements of Caspase-3-like Activity Spinal cords from E3.75 (st 22) to E5 (st 26) chick embryos were dissected in cold phosphate-buffered saline (PBS) (pH 7.4) and stored frozen at ⫺80°C. Spinal cord tissues were homogenized in 50 mM Tris–HCl (pH 7.4), 1 mM EDTA, 10 mM EGTA, followed by three rounds of freeze and thaw. After the addition of 10 ␮M digitonin, the tissues were incubated at 37°C for 10 min. Lysates were clarified by centrifugation at 15,000g for 3 min, and cleared lysates containing 5–10 ␮g protein were used for caspase measurements. Because no cell death occurs in the thoracic region at the ages examined here, thoracic spinal cord was used as a negative control. Assays were carried out in 96-well microtiter plates at 37°C for 1 h. The solution contained 100 ␮l of assay buffer (50 mM Tris–HCl (pH 7.4), 1 mM EDTA, 10 mM EGTA, and 1 mM DTT), lysates (5–10 ␮g protein), a final concentration of 50 ␮M substrate (Ac-DEVDMCA), and a final concentration of 50 ␮M inhibitor (Ac-DEVD-CHO). The production of 7-amino-4-methylcoumarin (AMC) from the enzyme-catalyzed cleav-

age of Ac-DEVD-MCA was measured using an excitation wavelength of 380 nm and emission wavelength of 460 nm on a CytoFluor II (Packard Bell). One unit was defined as the amount of enzyme required to release 0.22 nmol AMC per minute at 37°C.

Detection of DNA Fragmentation For detection of DNA fragmentation, the TUNEL reaction was used (Gavrieli et al., 1992). A reaction solution, composed of 1 mM CoCl 2, 50 ␮g/ml of gelatin, 5 nmol/ml of biotin-16 – dUTP (Boehringer MannheimYamanouchi), 50 U/ml of terminal deoxynucleotidyl transferase (Takara Shuzou), and 100 mM sodium cacodylate buffer (pH 7.0), was applied to the sections for 1 h at 37°C. Streptavidin–Texas Red conjugate or ABC kit (Vector) was used for detection of incorporated biotin-16 – dUTP.

Immunohistochemistry The monoclonal antibody 4D5, which recognizes the MN-specific antigen Islet-1, was obtained from the Developmental Studies Hybridoma Bank (Department of Biological Sciences, University of Iowa, Iowa City, IA). The supernatant of the hybridoma was diluted at 1:200 and applied to cryostat sections for 1 h at room temperature. Biotin-labeled anti-mouse IgG was applied for 1 h, and then streptavidin–Texas Red conjugate or ABC solution for 1 h. For peroxidase reaction, DAB was used as chromogen. A cleavage site-directed antibody against caspase-3 (anti-p20/17) was raised in rabbit as described previously (Kouroku et al., 1998; Urase et al., 1998). The antibody was diluted at 1:200 and applied overnight at 4°C. After washing, the FITC-conjugated anti-rabbit IgG was applied for 1 h. The anti-c-Jun/ AP-1 antibody (sc-45), which is now known to recognize the apoptosis-specific protein (ASP), was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The antibody was diluted at 1:2000 and applied to cryostat sections overnight at 4°C. After washing, the FITC-conjugated anti-rabbit IgG was applied for 1 h. For epifluorescent microscopy, sections were coverslipped with Vectashield containing DAPI (Vector). For peroxidase reaction, DAB was used as chromogen. After the reaction, sections were dehydrated and coverslipped with Bioleit (Ohken, Tokyo). Sections were observed and photographed on a Zeiss Axioskop microscope with an epifluorescent attachment and a Leica DC200 CCD camera unit.

180 In Vivo Administration of Caspase Inhibitors Ac-DEVD-CHO was dissolved into PBS immediately before use. BAF was first dissolved into dimethyl sulfoxide (DMSO) and then diluted with PBS. To avoid toxicity, the final concentration of DMSO was less than 2%. For dose–response experiments, these solutions were diluted to final concentrations. Treatment began when embryos reached stage 23(⫺). Windows were made in the shell over the embryo and to reduce the in ovo dilution of agents, 5 to 10 ml of egg white was removed with a syringe. Each dose was then administered directly onto the embryo. One-half of the original dose was subsequently administered at 6-h intervals. In some embryos, both BAF (200 ␮g) and Ac-DEVD-CHO (200 ␮g) were administered together, and in other embryos BAF (300 ␮g) was administered and one-quarter of this original dose was subsequently administered at 3-h intervals. After 12 or 24 h, when embryos had reached st 24 or st 25⫹/26⫺, respectively, they were moved from the shell into saline. The lower cervical region was dissected and divided along the midline into right and left halves. The 8th to 11th cervical segments were identified by dorsal root ganglia and processed for quantitative analyses of pyknosis and DNA fragmentation. The left side was placed in Bouin’s fixative overnight and embedded in paraffin. Serial paraffin sections were cut on a rotary microtome at 8 ␮m and stained with hematoxylin and eosin. The right side of C8 –C11 was fixed in 4% paraformaldehyde in 0.1 M phosphate buffer overnight and embedded in OCT compound. Frozen sections were cut on a cryostat at 7 ␮m and processed for the TUNEL reaction and immunohistochemistry. The C12 segment was processed for ultrastructural analysis. The left side of C12 was fixed overnight in 3% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3) at 4°C and then osmificated, dehydrated through a graded ethanol series, and embedded in Epon 812 (TAAB). Thin sections were cut on an ultramicrotome, stained with uranyl acetate and lead citrate, and examined with a JEOL-1200X electron microscope. The right side of C12 was fixed in 4% paraformaldehyde in 0.1 M phosphate buffer overnight and then embedded in Epon 812. Semithin sections of 1 to 2 ␮m were cut on an ultramicrotome and collected onto glass slides. Resin was removed by sodium methoxide processing (Apte and Puddle, 1990) and the sections were stained with HE.

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Quantification of Pyknosis, TUNEL-Positive Cells, Islet-1-Positive Cells, and Colocalization of ASP and Islet-1 The numbers of pyknotic cells and TUNEL-positive cells in the ventral horn region were counted in every fifth section of the C10 segment and the mean values were obtained. Because neurons dying following caspase inhibition exhibited a modified degenerating morphology (see Results), we have included these cells as well as typical pyknotic cells in our counts. In the case of BAF treatment, apparent degenerating cells exhibited dark (eosinophilic) HE staining and reduced nuclear size (⬍17.5 ␮m 2) and these criteria were used for quantification on E4.5 (see Results). For measuring nuclear size, the outlines of the nuclei of the MNs in the HE-stained 8-␮m-thick sections were drawn using an oil-immersion objective (⫻100) and drawing tube. Areas of the nuclei were measured by NIH Image software. The number of Islet-1-positive cells was counted in every 10th cryostat section of the C8 –C11 segments. For evaluation of the colocalization of ASP and Islet-1, simultaneous two-channel (FITC and Texas Red) confocal images were obtained at 1-␮m intervals using Olympus FluoView confocal laser microscopy. Colocalization was confirmed on the images and the number of such cells was counted.

ACKNOWLEDGMENTS The authors thank Drs. C. Milligan, M. Miura, H. Ogura, D. Prevette, N. Sato, S. Homma, and K. Isahara for their help and valuable advice and C. Sakuma, H. Ohuchi, T. Nakamura, M., Seino, and N. Kikuchi for their unfailing technical assistance. The monoclonal antibody 4D5, developed by Dr. T. Jessell, was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of NICHD and maintained by the Department of Biological Sciences, University of Iowa (Iowa City, IA). This study was supported by Grant-in-Aids for Scientific Research on Priority Areas and for Scientific Research from the Ministry of Education, Science, Sports, and Culture and Japan Society for the Promotion of Science (08680808, 10680704) to H.Y. and NIH Grant NS20402 to R.W.O.

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