Programmed cell death of developing mammalian neurons after genetic deletion of caspases

Abstract

An analysis of programmed cell death of several populations of developing postmitotic neurons after genetic deletion of two key members of the caspase family of pro-apoptotic proteases, caspase-3 and caspase-9, indicates that normal neuronal loss occurs. Although the amount of cell death is not altered, the death process may be delayed, and the cells appear to use a nonapoptotic pathway of degeneration. The neuronal populations examined include spinal interneurons and motor, sensory, and autonomic neurons. When examined at both the light and electron microscopic levels, the caspase-deficient neurons exhibit a nonapoptotic morphology in which nuclear changes such as chromatin condensation are absent or reduced; in addition, this morphology is characterized by extensive cytoplasmic vacuolization that is rarely observed in degenerating control neurons. There is also reduced terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling in dying caspase-deficient neurons. Despite the altered morphology and apparent temporal delay in cell death, the number of neurons that are ultimately lost is indistinguishable from that seen in control animals. In contrast to the striking perturbations in the morphology of the forebrain of caspase-deficient embryos, the spinal cord and brainstem appear normal. These results are consistent with the growing idea that the involvement of specific caspases and the occurrence of caspase-independent programmed cell death may be dependent on brain region, cell type, age, and species or may be the result of specific perturbations or pathology.

Fig. 1.

The spinal cord and brainstem of caspase-3-deficient mice appear morphologically normal. Nissl-stained transverse sections of postnatal day 10 (P10) mouse spinal cord (A–H) and brainstem–facial motor nucleus (I–L) of caspase-3-deficient (A, B, E, F, I, J) and control animals (C, D, G, H, K, L) are shown. A–D, Brachial;E–H, lumbar; I–L, brainstem–facial nucleus. Scale bars: A, C, E, G, 300 μm (shown inA); B, D, F, H, 50 μm (shown inB); I, K, 250 μm (shown inI); J, L, 120 μm (shown inJ). Dotted lines indicate ventral horns; an arrow indicates the facial motor nucleus.

Fig. 2.

Nissl-stained transverse sections of E16.5 lumbar spinal cord of homozygous littermate control (+/+) (A) and caspase-9-deficient −/− (B) embryos. Dotted lines inA and B indicate the medial border of the ventral horn. Scale bar, 100 μm. C, E, G, I, Degenerating neurons (arrows) from E14.5 caspase-3 (C, E) and caspase-9 (G, I) control (+/+) embryos. Note fragmented apoptotic bodies inG and I. Scale bar, 10 μm. D, F, H, J, Degenerating neurons (arrows) from caspase-3-deficient (D, F) and caspase-9-deficient (H, J) (−/−) embryos. Note the lack of prominent pyknosis and apoptotic bodies and the increased cytoplasmic density (compare G andH) in caspase-deficient dying neurons.Asterisks indicate normal non-dying cells.

Fig. 3.

Transverse sections through the middle of the superior cervical ganglion (SCG) of homozygote (A) and caspase-3 KO (B) littermates at P8. The overall size and organization of the ganglia appear normal in both low-magnification (insets) and high-magnification photomicrographs from KO animals.Arrows indicate some of the SCG neurons; double arrows indicate non-neuronal cells. Scale bars: A, B at full size, 25 μm; insets, 40 μm.

Fig. 4.

Photomicrographs of degenerating spinal cord neurons from E14.5 caspase-3 (Casp3) +/+ (A, C, E) and caspase-3 (Casp3) KO −/− (B, D, F) littermate embryos showing the distinct morphology of dying neurons in the caspase KO. These cells exhibit reduced chromatin condensation and nuclear pyknosis, marked cytoplasmic vacuolization, and dilation of mitochondria and RER compared with neurons from the caspase-3 +/+ embryos. C, Cytoplasm/soma;N, nuclei. Asterisks indicate normal non-dying neurons. The arrow in Aindicates the cytoplasm, the arrows in Eand F indicate mitochondria, and thearrowheads in D indicate the cell boundary of this degenerating motoneuron. Note the numerous vacuoles and abnormal organelles in the cytoplasm of the cells inD and F. Only part of the cytoplasm of a dying cell is shown in E. The scale bars inA, C, and E are the same for B, D, and F, respectively.

Fig. 5.

The number (mean ± SD) of degenerating MNs in the lumbar spinal cord of control +/+ (CON), caspase-3 −/− (Casp3−/−), and caspase-9 −/− (Casp9−/−) embryos on E14.5 and E16.5 expressed as the number per 1000 surviving MNs. The sample size (number of embryos) is indicated in thebars. In all groups, there was a significant (p < 0.01) reduction between E14.5 and E16.5.

Fig. 6.

Caspase-3 immunocytochemistry (A, B), TUNEL (C, E), and bisbenzimide staining (D, F) in E14.5 wild-type (A, C, D) and caspase-3 KO (B, E, F) lumbar spinal cord. A, Immunocytochemistry shows the staining of a cleaved (activated) caspase-3 subunit in the spinal motor neuron column and spinal ganglia in E14.5 wild-type mouse spinal cord (arrowheads), indicating the activation of caspase-3. B, In contrast, no staining of activated caspase-3 subunit is found in E14.5 caspase-3-deficient spinal cord. However, when the TUNEL method is used to detect cell death (C, E), it reveals positive staining in both wild-type (C) and caspase-3-deficient (E) spinal cord, although the staining patterns are distinct. The TUNEL-positive profiles in C andE represent the nucleus of an individual neuron. In wild-type spinal cord, there are more TUNEL-positive nuclei showing punctuate nuclear staining (C), which is correlated with condensed chromatin as revealed by bisbenzimide staining (D). In contrast, there are fewer TUNEL-positive cells in caspase-3-deficient spinal cord (see Results). Moreover, these cells typically show a distinct kind of TUNEL staining (E) and no clear evidence of chromatin condensation as indicated by the size of TUNEL-positive nuclei and reduced bisbenzimide-stained chromatin (F). Although not obvious in this photomicrograph, the nucleus indicated by the arrow inF is weakly labeled with bisbenzimide, indicating little, if any, chromatin condensation. Scale bars: A, B, 400 μm; C–F, 20 μm.