Evidence that Wallerian degeneration and localized axon degeneration induced by local neurotrophin deprivation do not involve caspases - PubMed (original) (raw)
Evidence that Wallerian degeneration and localized axon degeneration induced by local neurotrophin deprivation do not involve caspases
J T Finn et al. J Neurosci. 2000.
Abstract
The selective degeneration of an axon, without the death of the parent neuron, can occur in response to injury, in a variety of metabolic, toxic, and inflammatory disorders, and during normal development. Recent evidence suggests that some forms of axon degeneration involve an active and regulated program of self-destruction rather than a passive "wasting away" and in this respect and others resemble apoptosis. Here we investigate whether selective axon degeneration depends on some of the molecular machinery that mediates apoptosis, namely, the caspase family of cysteine proteases. We focus on two models of selective axon degeneration: Wallerian degeneration of transected axons and localized axon degeneration induced by local deprivation of neurotrophin. We show that caspase-3 is not activated in the axon during either form of degeneration, although it is activated in the dying cell body of the same neurons. Moreover, caspase inhibitors do not inhibit or retard either form of axon degeneration, although they inhibit apoptosis of the same neurons. Finally, we cannot detect cleaved substrates of caspase-3 and its close relatives immunocytochemically or caspase activity biochemically in axons undergoing Wallerian degeneration. Our results suggest that a neuron contains at least two molecularly distinct self-destruction programs, one for caspase-dependent apoptosis and another for selective axon degeneration.
Figures
Fig. 1.
Confocal immunofluorescence micrographs showing that procaspase-3 and its close relatives are not activated in RGC axons undergoing Wallerian degeneration in optic nerve explants (a) but are activated in RGCs undergoing apoptosis in retinal explants (b).a, Longitudinal sections of freshly dissected P7 optic nerve (0 hr) or P7 optic nerve explants cultured for 14, 19, or 24 hr were stained with the N52 anti-neurofilament antibody or with antibodies that specifically recognize either the activated form of caspase-3 (CM1 and Ab206) or proteins that have been cleaved by caspase-3 or its close relatives (Ab127). Note the lack of activated caspase-3 in nerves undergoing Wallerian degeneration, even though neurofilament degradation is apparent. Arrows point to apoptotic glial cells containing activated caspase-3 or its close relatives, which served as positive controls for our staining procedure. b, Cross sections of freshly dissected P7 retinae (0 hr) or P7 retinal explants cultured for 24 hr were stained with PI to visualize nuclei or with the CM1, Ab206, or Ab127 antibodies. Arrows point to the RGC layer. PI and CM1 staining are shown for the same fields. Note the single RGC undergoing naturally occurring apoptosis that was recognized by Ab206 at 0 hr. Scale bar, 50 μm.
Fig. 2.
Confocal immunofluorescence micrographs showing that procaspase-3 and its close relatives are not activated during Wallerian degeneration of sciatic nerve. Longitudinal sections of uncut sciatic nerve (0 hr) or sciatic nerve explants cultured for 14, 19, or 24 hr were stained with the N52 anti-neurofilament antibody and with the CM1, Ab206, or Ab127 antibodies. Note the lack of activated caspase-3 in nerves undergoing Wallerian degeneration, even though neurofilaments have been degraded. Arrows point to apoptotic glial cells containing activated caspase-3 or its close relatives. Scale bar, 50 μm.
Fig. 3.
Caspase activity in cytosolic extracts of sciatic nerve and thymocytes. Extracts were prepared from P0 thymocytes treated with STS and CHX (Thy + STS) or from P7 sciatic nerves that were cultured as explants for various times or that were treated with STS and CHX for 4 hr (4 h + STS) or 10 hr (10 h + STS). The extracts were assayed for their ability to cleave the fluorogenic caspase substrate zDEVD-AFC. There is little caspase activity in extracts from nerves undergoing Wallerian degeneration. Data from a representative experiment are shown. The experiment was repeated three times with similar results, and similar results were obtained when zVEID-AFC or zYVAD-AFC was used as a substrate.
Fig. 4.
Confocal immunofluorescence micrographs of sections of P7 optic nerves (a) and retinae (b) showing that caspase inhibitors inhibit apoptosis of axotomized RGCs but not Wallerian degeneration of their axons. Explants were cultured for 24 hr in the presence of the caspase inhibitor zVAD-fmk or the cathepsin B inhibitor zFA-fmk.a, Longitudinal sections of optic nerve were stained with the N52 anti-neurofilament antibody. Note that neither inhibitor prevented the breakdown of neurofilaments. _b, Left,Cross sections of retina were stained with propidium iodide. The_arrowheads point to the RGC layers. Note that pyknotic RGCs are apparent in explants treated with zFA-fmk but not in those treated with zVAD-fmk. Right, The bar graph shows the number of pyknotic (pyk.) cells per field in the RGC layer of the retinae (expressed as the mean ± SD of three experiments). Scale bar, 50 μm.
Fig. 5.
Differential interference contrast (DIC) and confocal immunofluorescence micrographs showing the effect of caspase inhibitors on transected DRG axons in culture. P0 DRG explants were cultured for 7d, treated with zVAD-fmk or the calpain inhibitor ALLN for 1 hr, and then axotomized. After 24 hr, the transected axons were fixed and then studied by _DIC_microscopy or stained with the N52 anti-neurofilament antibody and then studied with confocal immunofluorescence microscopy. Note that ALLN prevented the breakdown of some neurofilaments but not the axolemma, whereas zVAD-fmk had no effect on either. Different fields are shown for DIC images (scale bar, 33 μm) and immunofluorescence images (scale bar, 50 μm).
Fig. 6.
Confocal immunofluorescence micrograph of activated caspase-3 in apoptotic DRG neurons deprived of NGF. Dissociated P0 DRG neurons were cultured for 7 d in medium containing NGF and were then deprived of NGF and treated with anti-NGF antibodies for 24 hr. The cells were then fixed and stained with CM1 antibodies. Note that activated caspase-3 is present in the cell body and axons of the two neurons. The CM1 staining extended throughout the entire length of the axons (∼1 mm; data not shown). Scale bar, 50 μm.
Fig. 7.
DIC and immunofluorescence micrographs showing the effect of caspase inhibitors on axons locally deprived of NGF. P0 DRG neurons were dissociated and plated in the central compartment of a three-chambered culture dish, with NGF in all of the compartments. After 7 d, the neurons had extended axons into the two distal compartments, and the medium in some of the right-hand distal chamber was replaced with medium that lacked NGF and contained both anti-NGF antibodies and zVAD-fmk. Four days later, the neurons were fixed and then studied by DIC microscopy or stained with the N52 anti-neurofilament antibody and then studied with confocal immunofluorescence microscopy. Many of the axons in compartments deprived of NGF and treated with zVAD-fmk (shown) degenerated; axons in compartments deprived of NGF without zVAD-fmk (data not shown) showed comparable degeneration. Different fields are shown for DIC and immunofluorescence images. Scale bar, 100 μm.
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