Programmed Cell Death of Developing Mammalian Neurons after Genetic Deletion of Caspases

MATERIALS AND METHODS

Animals and histology. Heterozygous caspase-3 and caspase-9 mutant mice were maintained in a mixed C57BL/6 and 129sv strain background and were interbred to generate homozygous mutants for this study. Sibling animals at indicated embryonic stages were collected and individually genotyped by PCR as described previously (Kuida et al., 1996, 1998). Given the variable severity of caspase-3 and caspase-9 mutant phenotypes, only mutant embryos showing forebrain abnormalities (exencephaly) were included in our analysis. Because most caspase-9-deficient embryos die in utero after embryonic day 16.5 (E16.5) (Kuida et al., 1998), only E16.5 or younger animals were examined here. Most of the embryos and postnatal animals were immersion-fixed in either Bouins' or Carnoy's solution; the spinal cord (with attached DRG) and brain were processed separately, embedded in paraffin, sectioned serially (10 μm), and stained with either thionine or hematoxylin eosin (Li et al., 1994). Some animals were also fixed in 4% paraformaldehyde and 2% glutaraldehyde, post-fixed in 2% osmium tetraoxide, dehydrated in graded alcohol, and embedded in plastic (Epon); semiserial sections were stained with toludine blue. A polyclonal antibody specific for the 17 kDa cleaved subunit of caspase-3 (BD 559565; PharMingen, San Diego, CA) was used for in situ detection of caspase-3 activation. FITC-conjugated anti-rabbit IgG secondary antibody (Jackson ImmunoResearch, West Grove, PA) was used for visualization of the staining. Spinal cord sections stained with bisbenzimide for cell nuclei and with FITC-labeled activated caspase-3 were imaged using aZeiss LSM510 microscope (Zeiss, Thornwood, NY). Simultaneous confocal–two-photon excitation using an argon laser (488 nm) for FITC, and an infrared pulse laser (760 nm) for bisbenzimide was used to localize cells expressing the active form of caspase-3. For terminal deoxynucleotidyl transferase (TdT)-mediated biotinylated UTP nick end labeling (TUNEL) staining, 10 μm paraffin-embedded tissue sections were first rehydrated and permeabilized with 0.2% Triton X-100 before nick end-labeling with 0.32 U/μl TdT and 2 mmbiotin-16-dUTP (Boehringer Mannheim, Indianapolis, IN). Texas Red-conjugated streptavidin (PerkinElmer Life Sciences, Emeryville, CA) was used for visualization of the TUNEL-positive signals. The number of TUNEL-positive neurons was counted in an equal number of sections spanning 470 μm in two wild-type (WT) and two caspase-3 knock-out (KO) lumbar spinal cords on E14.5.

Morphometric analysis. Neurons were identified and counted according to criteria described by Clarke and Oppenheim (1995), who indicated that reliable and accurate cell counts can be attained without the use of correction factors or stereological techniques. For each neuronal population examined, all cells that met these criteria were counted in every fifth or 10th section, and these totals were multiplied by 5 or 10 as an estimate of the total number of neurons in that population. Because neurons dying in the absence of caspase-3 or caspase-9 exhibit a modified degenerating morphology when examined using the light microscope (see Results), we have included these cells as well as typical pyknotic cells in our counts of degenerating neurons. Counts of surviving and dying neurons in the intermediate gray matter of the lumbar spinal cord excluded the dorsal and ventral horns, and cells were counted on only one side (hemisection) in every 10th section. The degeneration data from caspase-3 and caspase-9 embryos (control and mutant) were similar and thus have been pooled for further analysis. Finally, because we have never observed any differences in neuronal numbers between caspase-3 or caspase-9 homozygotes (+/+) and heterozygotes (+/−), these cell counts have been pooled and compared with caspase-3- and caspase-9-deficient (−/−) littermates.

RESULTS

DISCUSSION

Caspases are considered to be one of the most highly conserved and ubiquitous families of pro-apoptotic molecules involved in PCD. In the nematode, CED-3 and CED-4 are required for virtually all developmental cell deaths (Ellis et al., 1991; Horvitz, 1994), and in vertebrates, interactions of their counterparts (caspase-3, caspase-9, and Apaf-1) are also thought to be essential for the PCD of many cell types, especially neurons. Null mutant mice lacking each of these genes exhibit a remarkably similar CNS malformation (exencephaly) that has been attributed in part to a failure of apoptotic PCD of neurons (Kuida et al., 1996, 1998; Cecconi et al., 1998; Hakem et al., 1998; Yoshida et al., 1998; Roth et al., 1999). This explanation of the exencephaly is primarily based on direct evidence for decreased TUNEL-positive and pyknotic cells and increased numbers of mitotically active cells in the forebrain (Yoshida et al., 1998). However if TUNEL or a typical pyknotic morphology is not always a reliable marker for PCD in these mutants, then it is possible that it is the specific kind of DNA degradation detected by these methods, but not cell death per se, that is reduced or absent in the mutants. Accordingly, it seems likely that other aspects of CNS development may be abnormal (e.g., proliferation) and may contribute to the exencephaly in the forebrain. Although the cellular mechanisms are not well understood, a failure of neural fold elevation appears to be involved in virtually all cases of exencephaly in mice (Harris and Juriloff, 1999).

There is increasing evidence that TUNEL is not a reliable index of PCDin vitro for either neuronal or non-neuronal cells that lack caspase-3 (knock-outs) or after reductions in caspase-3 or other caspases by treatment with peptide inhibitors (Xiang et al., 1996;Jänicke et al., 1998a,b; Stefanis et al., 1998, 1999; Weil et al., 1998; Woo et al., 1998; Zheng et al., 1998; Bortner and Cidlowski, 1999; Cregan et al., 1999; Ferrer, 1999; Xue et al., 1999;D'Mello et al., 2000; Keramaris et al., 2000; Sperandio et al., 2000). The dying cells in these cases are reported to degenerate in the absence of the typical nuclear changes that are considered to be hallmarks of apoptosis, including pyknosis and the DNA degradation recognized by the TUNEL technique. Although the death of cells in this situation is reported to be delayed (Cregan et al., 1999; Keramaris et al., 2000; Williams et al., 2000), they exhibit degradative changes in the cytoplasm when they do degenerate, and these changes appear to be distinct from those occurring during classic apoptosis, including extensive vacuolation (Xiang et al., 1996; Woo et al., 1998;Sperandio et al., 2000).

Although our results suggest that caspase-3 is normally involved in the PCD of developing postmitotic neurons in vivo, data from caspase-3- and caspase-9-deficient animals, together with other evidence (Shiraiwa et al., 2001), clearly indicates that in the absence of these caspases the extent of cell death in many neuronal populations is normal. However, the mode of degeneration includes reduced TUNEL and nuclear and cytoplasmic changes that are distinct from classic apoptosis. Because the neuronal populations examined here by us all undergo PCD as postmitotic cells, it is interesting that we have not observed the kind of malformations (exencephaly, spina bifida) in the spinal cord or hindbrain that occur in more rostral, mitotically active regions of the brain of caspase-3- and caspase-9-deficient mice. This is consistent with the suggestion that it may be primarily the delay in PCD and the subsequent increased proliferation of these rostral forebrain precursor cells that is responsible for the forebrain abnormality (Kuan et al., 2000). Although additional detailed studies of the kinetics of neuronal degeneration in caspase-3- and caspase-9-deficient embryos are needed to determine whether there is in fact a delay, as occurs in caspase-3-deficient cultured cells (D'Mello et al., 2000) and in chick embryo neurons in vivo (Shiraiwa et al., 2001), the evidence presented here for spinal interneurons is consistent with this possibility. If there is in fact a delay in the kinetics of cell death in the mutant E16.5 embryos, it is more likely to be detected in spinal interneurons, which are at the beginning of their normal period of cell death on E16.5 (Lawson et al., 1997;Grieshammer et al., 1998), compared with MNs, which are near the end of their cell-death period at this age (Oppenheim et al., 1986) and therefore may have had sufficient time to undergo even a delayed cell death. A delay in the degeneration of caspase-deficient neurons suggests that caspase-mediated neuronal death is a more efficient means of PCD than caspase-independent PCD.

We have also observed that the extent of PCD of spinal and cranial motoneurons and DRG sensory cells is normal in Apaf-1-deficient embryos (Oppenheim et al., 2001), consistent with the report that the hindbrain and spinal cord in these mutants appear normal (Cecconi et al., 1998). This raises the question of what molecular pathways are being used in place of Apaf-1, caspase-9, and caspase-3 for mediating PCD in these neuronal populations. One possibility is that these neurons have the capacity to switch to another, caspase-independent, degradative pathway of PCD. Interestingly, cultured PC12 cells and peripheral sensory neurons in which caspase activity is inhibited do in fact switch to a death-inducing pathway that uses lysosomal proteases (cathepsins) instead of caspases for PCD, and they exhibit a morphology similar to that described here for caspase-3- and caspase-9-deficient embryos (Isahara et al., 1999; Agerman et al., 2000). It is also possible that neuronal death in these situations involves the recently described caspase-independent pathway that uses the apoptosis-inducing factor which activates an apoptotic-like mode of degeneration (Joza et al., 2001). Alternatively, other caspases may be able to compensate for the actions of caspase-3 and caspase-9 in mutant mice (Cryus and Yuan, 1998; Hakem et al., 1998; Stefanis et al., 1998; Woo et al., 1998; Zheng et al., 1998, 2000). It has been reported recently that cultured mammalian MNs may use Fas receptors and caspase-8 instead of caspase-9 during PCD (Raoul et al., 1999). However, because caspase-3 is thought to be the major downstream caspase in the Fas pathway, this mode of cell death would not be available for compensation in the caspase-3 KO animals. Cultured NGF-deprived sympathetic neurons treated with the pan-caspase inhibitor bok–asp–fmk (BAF) also continue to undergo cell death, albeit by an apparent autophagic (Clarke type 2), nonapoptotic pathway (Xue et al., 1999). The dying neurons in the chick embryo after caspase inhibition by BAF (Shiraiwa et al., 2001) and those observed here in caspase-deleted mice do not appear to be undergoing an autophagic cell death because the cytoplasmic vacuoles in these cases appear to be empty of lysosomal material. Rather, we suggest that cell death in this situation more closely fits the type 3B “cytoplasmic” mode of embryonic neuronal death reported by Clarke (1990) during normal development. The cytoplasmic vacuoles observed in type 3B PCD appear to reflect the extreme dilation and swelling of cytoplasmic organelles, including mitochondria (Pilar and Landmesser, 1976; Chu-Wang and Oppenheim, 1978). In some respects, dying cells in caspase-deficient animals resemble necrosis (Hirsch et al., 1997; Lemaire et al., 1998). However, consistent with several other studies, we consider this form of degeneration to be a nonapoptotic form of normal PCD (Pilar and Landmesser, 1976; Chu-Wang and Oppenheim, 1978; Clarke, 1990; Sperandio et al., 2000). In a recent study of thymocyte apoptosis, it was shown that during phagocytosis, macrophage-derived lysosomal proteases can induce cell death and DNA fragmentation in thymocytes by a caspase-independent, cell-nonautonomous pathway (McIlroy et al., 2000). Studies are in progress to more fully characterize the mechanisms involved and the morphological changes that occur in degenerating avian and mouse neurons after the loss of caspases.

We find it rather remarkable that in the absence of two of the major pro-apoptotic caspases thought to be required for neuronal cell death, not only do neurons in the diverse populations examined here continue to undergo PCD, but they do so in a highly regulated manner such that the same proportion of cells undergo PCD as do cells in the presence of caspase-3 or caspase-9. Although we assume that all of these populations undergo normal PCD in caspase-9-deficient animals, because we have not been able to examine embryos older than E16.5, we cannot entirely exclude the possibility that later stages of PCD may not be normal. Because this seems unlikely, however, our results indicate that the control of cell number in these neuronal populations by cell death is regulated at a point upstream of the specific machinery used for actually killing the cell. Although we have not examined the release of cytochrome c during the degeneration of caspase-deficient neurons, several studies have shown that caspase deficiency does not affect cytochrome c release in dying sympathetic, cortical, and motor neurons in vitro (Desmukh et al., 2000; Keramaris et al., 2000; Li et al., 2001).

In summary, many populations of developing postmitotic mammalian neurons are able to exhibit normal amounts of PCD in the absence of either caspase-3 or caspase-9. However, the morphology of these degenerating neurons differs from the more typical apoptotic type of cell death and the kinetics of their degeneration may be delayed. These data, together with several other lines of evidence reviewed here, demonstrate that the use of nuclear changes alone for assessing PCD can often be misleading. For example, the apparent discrepancy between the report by Miller et al. (1997) and that by Eldadah et al. (2000)regarding the role of caspase-3 in the death of cultured cerebellar granule neurons may be explained by a primary reliance on nuclear changes alone on the one hand (Eldadah et al., 2000) versus the use of nuclear changes as well as other markers on the other hand (Miller et al., 1997; D'Mello et al., 2000) for assessing the death of neurons. Similarly, whereas the use of TUNEL indicates a significant reduction in the PCD of DRG cells in E12.5 caspase-9-deficient embryos (Zaidi et al., 2001), we find no differences in the number of surviving DRG cells in these mutants on E16.5. Together, these and other recent reports (see the introductory remarks) as well as our own in vivoobservations here and elsewhere (Shiraiwa et al., 2001) support the growing idea that distinct caspases as well as other caspase-independent pathways may mediate neuronal death in specific brain regions and cell types, at specific ages, in different species, and after particular kinds of perturbations (Agerman et al., 2000; Kuan et al., 2000; Zaidi et al., 2001). Finally, these data suggest a note of caution in attempts to use caspase inhibition as a therapeutic strategy for preventing cell death in neurodegenerative diseases (Kermer et al., 1999; Schulz et al., 1999), because compensatory mechanisms for cell death may be activated when caspase-dependent pathways are blocked.