A caspase cascade regulating developmental axon degeneration - PubMed (original) (raw)

Figure 1.

Figure 1.

Loss of Caspase-6 partially protects against degeneration induced by NGF withdrawal. A, Wild-type (WT) and _Caspase-6_-knockout (KO) DRGs were cultured in Campenot chambers for 7 days in 50 ng/ml NGF and subsequently deprived of NGF for 36 and 60 h by washing out NGF and adding a function blocking anti-NGF antibody. Representative images of three rows of axons per chamber, per condition, in the presence or absence of NGF at 36 h. B, Axon degeneration was quantified as the percent of total axons with fragmented staining of βIII-Tubulin (TuJ1) across all wells of the chamber. To exclude the possibility that loss of Caspase-6 (or other mutations used in this study) affects the number of axons that traverse the grease barrier, we quantified the number of axons in the control (+NGF) side of each chamber and found no significant difference (data not shown). Each chamber derived from a unique embryo. n = 11 chambers per genotype; *p = 0.0130 at 36 h time point, Student's t test.

Figure 2.

Figure 2.

Processing of zymogen Caspase-6 by active Caspases-3 and -6. A, Domain organization of zymogen Caspase-6 highlighting the catalytic C163A mutation and the three cleavage sites. B, Processing of zymogen Caspase-6 by a panel of catalytically active caspase family members showing different extents of cleavage by Caspases-3 and -6. The band observed in lane 2 corresponds to the large subunit of Caspase-2. C, D, Zymogen Caspase-6 is fully processed by active Caspase-3 in a concentration-dependent manner. E, F, Zymogen Caspase-6 is efficiently processed in trans only at its prodomain by active Caspase-6. The cleavage products from processing by Caspase-3 (Casp-3) or Caspase-6 (Casp-6) were confirmed by N-terminal sequencing (data not shown) and quantified, and they are plotted in D and F, respectively. G, Domain organization of zymogen Caspase-6 highlighting the catalytic C163A, and D23A/D179A mutations. A point mutation (D186A) was also introduced to prevent potential cleavage in the middle of the linker region. H, I, Zymogen Caspase-6/D23A /D179A mutant is processed weakly by active Caspase-6 but not Caspase-3.

Figure 3.

Figure 3.

Caspase-3 is required for axon degeneration following NGF withdrawal. A, E12.5 DRG explants from wild-type (WT) and Caspase-3 knockout (KO) mice were cultured for 2 days and subsequently deprived of NGF for 24 h using a function blocking antibody. WT explants had completely degenerated while we observed no apparent degeneration in the Caspase-3 KO. B, Identical findings were seen in Campenot chambers. Representative images are shown of three rows of axons per genotype. WT degeneration is shown after 36 h in anti-NGF, while the Caspase-3 KO cultures are shown following 60 h in anti-NGF to illustrate the magnitude of the protection observed. C, Quantification of phenotype from B. Axons are significantly protected from NGF deprivation at both 36 and 60 h; n = 6 Campenot chambers per genotype per time; *p < 0.001 at each time point, Student's t test. D, We observed protection against anti-NGF in Campenot chamber cultures treated with 30 μ

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z-DEVD-Fmk when the axons were from Caspase-3 heterozygous (Het) mice, whereas no protective effect was observed in WT axons. Representative images from the 36 h time point. E, Quantification of D. Significant protection is observed at the 36 h time point (p < 0.001, Student's t test, n = 5 per condition) but not at 60 h. Axons are visualized by TuJ1 immunoreactivity (A, B, D). F, Cleavage of Caspase-6 is visualized by immunostaining in WT explants deprived of NGF for 12 h. This cleavage is eliminated in Caspase-3 knockout explants. G, No apparent protection from injury-induced Wallerian degeneration of axons was observed in Caspase-3 knockout explants following physical severing of proximal axons in vitro. Caspase-6 knockout explants were also not protected (data not shown). H, Similarly, loss of Caspase-3 does not protect axons in Campenot chambers from degeneration elicited by the chemotherapeutic agent vincristine.

Figure 4.

Figure 4.

Detection of cleaved Caspase-3 in NGF-deprived sensory axons. A, Wild-type (WT) and Caspase-3_-knockout (KO) DRG explants were cultured from E12.5 embryos in the presence of NGF for 2 days, fixed, and stained with either of two antibodies to pro-Caspase-3, a polyclonal (Millipore, catalog no. 06-735) and a monoclonal (Millipore, clone E87; data not shown). While diffuse axonal staining was observed, in neither case did this immunoreactivity disappear in the Caspase-3 knockout. B, Parallel cultures were lysed, either as full explants or when the cell bodies have been mechanically removed (see Materials and Methods, referred to as “Axon”). The purity of axonal protein preparations was confirmed by immunoblotting with the nuclear marker Lamin A/C. The Millipore polyclonal antibody detected a single band at the rough molecular weight of Caspase-3; however, this band did not disappear in the knockout. By contrast, the Cell Signaling Technology antibody (mAb 8G10) detected a band that disappeared in the knockout, indicating both the presence of pro-Caspase-3 and the specificity of this antibody, which was therefore used to detect activation of Caspase-3 in Figure 5_A. Several attempts to detect a signal by immunoblotting with mAb E87 were unsuccessful (data not shown), so, like the polyclonal antibody, E87 was not used for other experiments. C, WT axons were grown in Campenot chambers and subsequently deprived of NGF for varying times. Axons were fixed and stained with a neo-epitope antibody directed against the Asp175 cleavage site in Caspase-3. Staining for cleaved Caspase-3 is only seen in a subset of axons. Stainings were performed before overt TuJ1 fragmentation, here at 30 h following NGF withdrawal. In our experiments, fragmentation assessed by TuJ1 antibody in Campenot chambers begins rapidly after the 30 h time point, and axons are often 40–50% fragmented by 36 h (see Figs. 1_B_, 3_B_). D, DRG explants were grown for 2 or 7 days, and both cell bodies and axons were deprived of NGF for the indicated times. Axons from 7 day cultures took considerably longer to fragment, as assayed by TuJ1 staining. Similar to Campenot chambers grown for the same 7 day period, cleaved Caspase-3 staining was not readily detected in 7 day explant cultures deprived of NGF, but was readily detected when those same cultures were exposed to staurosporine (3 h, 1 μ

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, top). In contrast, in 2 day cultures strong and uniform cleaved Caspase-3 staining develops over time in and is eliminated in Caspase-3 knockout explants (bottom). Inset: Higher magnification examples of the punctate appearance of cleaved Caspase-3.

Figure 5.

Figure 5.

Cleavage of Caspase-3 is downstream of Caspase-9 and the anti-apoptotic Bcl-2 family. A, Western blot analysis of isolated axonal protein following a time course of NGF withdrawal. Caspase-3 is processed from its immature zymogen form into its smaller, active subunit, marked with an arrow. During this period we do not observe a change in XIAP levels. B, Wild-type (WT) DRG explants were incubated with an IAP antagonist (MV1, 5 μ

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) and stained with TuJ1. No apparent degeneration was observed over the 16 h exposure, nor with a panel of other IAP antagonists (data not shown). As a control for the efficacy of MV1 we performed Western blot analysis on parallel cultures and observed degradation of c-IAP1 with two separate antibodies, a hallmark of having reached an efficacious concentration (Varfolomeev et al., 2007). C, We observed strong staining for cleaved Caspase-9 in 9 h NGF-deprived DRG explants. D, A Caspase-9 knockout (KO) mouse was generated for the purpose of this study following the published methodology (see Materials and Methods) of removing the catalytic motif in exon 6. To confirm that Caspase-9 expression is lost, we performed Western blot analysis of lysates from E12.5 spinal cord. E, Genetic deletion of Caspase-9 blocked cleavage of Caspase-3 (visualized at 9 h) and axon degeneration (visualized at 24 h) in NGF-deprived explants. F, Application of the Bcl-2 antagonist ABT-737 (20 μ

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, 3 h) leads to Caspase-3 cleavage in 2 day DRG explants, and the pro-degenerative effects of ABT-737 (20 μ

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, 16 h) are suppressed in Bax and Caspase-3 KO cultures in Campenot chambers. G, Axons are visualized by TuJ1 immunoreactivity. H, Model for the regulation of axon degeneration in NGF deprived sensory neurons.

Figure 6.

Figure 6.

Impaired pruning of retinocollicular axons in Caspase-3 by DiI labeling. A, Superior colliculus from a P6 Caspase-3 heterozygous (P6 Casp Het) mouse 24 h after focal injection of the lipophilic axon tracer DiI into the periphery of temporal retina. B, SC from a Caspase-3 knockout (KO) P6 mouse 24 h after focal DiI injection into the periphery of temporal retina. In Caspase-3 KO mice, an appropriately sized TZ at the appropriate location is observed. In addition, RGC axon segments and rudimentary arbors are maintained in ectopic locations (arrows) posterior (P) and lateral (L) to the TZ. C, D, Vibratome sections (sagittal, 100 μm) through the SC of P6 mice injected as those depicted in A, B are shown. C, In Caspase-3 heterozygous mice very few axon segments persist away from the TZ. In addition, arbors are always directly associated with the TZ. D, In Caspase-3 KO mice, axon segments are evident well posterior to the TZ (arrows). In addition, rudimentary arbors are found in aberrant positions hundreds of microns lateral and posterior to the TZ (top and bottom panels). The bottom panel shows sections taken from the case in B from the lateral position of the left arrow. E, A grid of 100 μm square domains overlaid on a P6 SC. F, In Caspase-3 knockout P6 mice, axon segments persist in a significantly greater number of total 100 μm square domains outside of the dense TZ (47% increase, p < 0.05; Student's t test). This difference is more pronounced in regions more distant to the TZ (p < 0.05). Arrowheads designate the anterior border of the SC. M, Medial. Scale bar (in D), 300 μm.

Figure 7.

Figure 7.

Impaired pruning of retinocollicular axons in Caspase-3 and Caspase-6 by in utero labeling. A, Representative maximum intensity projection images of GFP-positive axons within the SC at postnatal days 1 P1 and P6. Yellow box indicates the PSC. B, Schematic of the left superior colliculus used for GFP-labeling experiments; A, Anterior; P, posterior; M, medial; L, lateral; TZ, termination zone; PSC, posterior superior colliculus defined as the posterior region within 100 μm of imaged edge. C, Representative images of the PSC in control animals at postnatal days 1, 3, 5, and 6. C, Control; WT, wild type; Het, heterozygous; KO, knockout. D, Quantification of the number of GFP-positive axons in the PSC as a function of developmental age (P1, 158.4 ± 10.8, n = 5; P3, 31.7 ± 10.3, n = 6; P5, 7.8 ± 3.5, n = 7; P6, 4.9 ± 1.1, n = 15). E, Representative images of the PSC in _Caspase-3_- and _Caspase-6_-knockout mice. F, Quantification of the number of GFP-positive axons in the PSC of Caspase-3 (WT, 5 ± 3.1, n = 4; Het, 6.6 ± 2.5, n = 10; KO, 19.8 ± 3.0, n = 9) and Caspase-6 mutants (WT, 3.25 ± 1.0, n = 5; Het, 13 ± 2.6, n = 9; KO, 25.6 ± 6.3, n = 9) at P6. C corresponds to a wild-type P6 control group included throughout. G, Quantification of the spread of GFP labeling along the anterior–posterior axis of the SC as an estimate of the size of the TZ (n = 17, 3, 10, 9, 5, 8, and 9, respectively, for listed genotypes). H, Quantification of the number of GFP-positive axons in the optic nerve tract (n = 11, 3, 9, 6, 4, 4, and 5, respectively, for listed genotypes). I, Axon number in the PSC normalized to TZ size. J, The number of axons in the PSC plotted per animal as a function of the number of labeled axons within the optic nerve tract. K, The number of axons in the PSC plotted per animal as a function of TZ size. L, Axon number in the PSC normalized to the number of labeled axons in the optic nerve tract. Caspase-3 KO and Caspase-6 KOs exhibit a significant increase in normalized axon density compared to littermate controls (p < 0.04). Bar graphs represent mean ± SEM; *p < 0.05; **p < 0.01.