Distinct pathways mediate axon degeneration during apoptosis and axon-specific pruning

Abstract

Neurons can activate pathways that destroy the whole cell via apoptosis or selectively degenerate only the axon (pruning). Both apoptosis and axon degeneration require Bax and caspases. Here we demonstrate that despite this overlap, the pathways mediating axon degeneration during apoptosis versus axon pruning are distinct. While Caspase-6 is activated in axons following nerve growth factor deprivation, microfluidic chamber experiments reveal that Caspase-6 deficiency only protects axons during axon-specific but not whole-cell (apoptotic) nerve growth factor deprivation. Strikingly, axon-selective degeneration requires the apoptotic proteins Caspase-9 and Caspase-3 but, in contrast to apoptosis, not apoptotic protease activating factor-1. Additionally, cell bodies of degenerating axons are protected from caspase activation by proteasome activity and X-linked inhibitor of apoptosis protein. Also, mature neurons restrict apoptosis but remain permissive for axon degeneration, further demonstrating the independent regulation of these two pathways. These results reveal insight into how neurons allow for precise control over apoptosis and axon-selective degeneration pathways, thereby permitting long-term plasticity without risking neurodegeneration.

Figure 1. Casp6 activation in axons is selective to NGF deprivation

(a) Wild type (WT) sympathetic neurons (5 DIV) were treated with the following conditions to induce axon degeneration, respectively: NGF deprivation (-NGF), 20 μM etoposide (DNA Damage), 2.5 μM tunicamycin (ER Stress), and 1 μM vinblastine (to induce microtubule destabilization). NGF-maintained neurons served as controls. Neurons were fixed and immunostained for tubulin upon axon beading and fragmentation. (b) Neurons were treated as described in (a) and immunostained for cleaved Casp3 and cleaved Casp6. Nuclei were labeled with Hoechst. Scale bar, 20 μm.

Figure 2. Casp6 is required for axon degeneration during axon pruning but not apoptosis

(a) WT and Casp6-deficient (Casp6 KO) neurons(5 DIV) were NGF-deprived for 24 hours. WT neurons maintained in NGF served as a control. Scale bar, 10 μm. (b) Microfluidic chamber model of global deprivation, which induces soma and axon degeneration, and local deprivation, which results in axon-selective degeneration. (c) WT and Casp6-deficient neurons were NGF-maintained, globally deprived, or locally deprived. Neurons were immunostained for tubulin and nuclei were labeled with Hoechst. Scale bar, 50 μm. (d) Quantification of axon degeneration for conditions shown in (c). Data represent the mean ± s.e.m. (n=3). P values were calculated using an unpaired t-test.

Figure 3. Axon-selective degeneration is Apaf-1-independent but requires Casp9 and Casp3

(a) Sympathetic neurons (5 DIV) from mice deficient for Bax, Apaf-1, Casp9, Casp6, and Casp3 were locally deprived in microfluidic chambers. NGF-maintained and locally deprived WT littermate neurons served as controls. Neurons were immunostained for tubulin and cleaved Casp6. (b) WT neurons were locally deprived for 24 hours and probed for cleaved Casp3. NGF-maintained neurons served as controls. (c) Quantification of axon degeneration for conditions shown in (a). (d) Quantification of maintained axon length over time during local deprivation for all conditions shown in (a). All data represent the mean ± s.e.m. (n=3). (e) Phase images of the same axons over a 36-hour timecourse of local deprivation demonstrate similar degeneration kinetics between WT and Apaf-1-deficient axons. All p values were calculated using an unpaired t-test. All Scale bars, 50 μm.

Figure 4. Local deprivation triggers cytc release in axons

Neurons (5 DIV) were NGF-maintained, globally deprived, or locally deprived in the presence of pan-caspase inhibitor (25 μM QVD) for 36 hours and probed for cytc and the mitochondrial marker Tom20. Quantification of the colocalization index (ImageJ) for cytc and Tom20 is shown below. Data represent the mean ± s.e.m. (n=3). Scale bar, 10 μm. All p values were calculated using an unpaired t-test.

Figure 5. The proteasome restrictscaspase activation to axons during axon-specific degeneration

(a) WT neurons were NGF-maintained or locally deprived in the absence (-) or presence (+) of the proteasome inhibitor Bortezomib. Neurons were immunostained for cleaved Casp6 and tubulin (inset). Scale bar, 50 μm. Quantification of axon degeneration is shown on the right. (b) Cell bodies of neurons treated as described in (a) were immunostained for cleaved Casp3. Nuclei were labeled with Hoechst. Scale bar, 20 μm. Quantification of cleaved Casp3-positive cell bodies is shown on the right. (c) Cell bodies of neurons treated as described in (a) were immunostained for cleaved Casp9 and nuclei were labeled with Hoechst. Scale bar, 20 μm. Quantification of cleaved Casp9-positive cell bodies is shown on the right. (d) WT and Casp3-deficient neurons were locally deprived with (+) and without (-) Bortezomib and probed for tubulin. NGF-maintained neurons served as controls. Scale bar, 50 μm. Quantification of axon degeneration is shown on the right. All data represent the mean ± s.e.m. (n=3). All p values were calculated using an unpaired t-test.

Figure 6. XIAP protects the soma from caspase activation during axon-specific degeneration

(a)WT or XIAP-deficient neurons were locally deprived and probed for cleaved Casp6. Arrows indicate early signs of axonal collapse. Scale bar, 50 μm. Quantification of axon degeneration is shown on the right. (b) WT and XIAP-deficient neurons were NGF-maintained or locally deprived and cell bodies were probed for cleaved Casp3. Nuclei were labeled with Hoechst. Scale bar, 20 μm. Quantification of the percentage of cell bodies positively labeled for cleaved Casp3 is shown on the right. All data represent the mean ± s.e.m. (n=3). All p values were calculated using an unpaired t-test.

Figure 7. Mature neurons inhibit apoptosis during global deprivation but degenerate axons during local deprivation

(a) Young (5 DIV) and mature (28 DIV) sympathetic neurons were either NGF-maintained or NGF-deprived for 48 hours. Scale bar, 20 μm. (b) The same axons of young and mature neurons were imaged every 24 hours during local deprivation. NGF-maintained neurons at both ages served as controls. Scale bar, 50 μm. (c) Quantification of axon degeneration for conditions shown in (b). Data represent the mean ± s.e.m. (n=3). (d) Cleaved Casp6 immunostaining and corresponding phase images (inset) in the axon compartments of conditions shown in (b). All p values were calculated using an unpaired t-test.

Figure 8. Working model of the pathways mediating apoptosis versus axon pruning

Proteins highlighted in yellow have been shown to be required for both apoptosis and axon pruning pathways. Our results indicate that Apaf-1 and Caspase-6 (highlighted in blue) are two points of divergence. Future experiments will be required to determine what role, if any, is played by the mitochondria and cytc release in axon-selective degeneration. In addition, how Caspase-9 is able to activate Caspase-6 and Caspase-3 without Apaf-1 remains to be determined. While Casp6 and Casp3 are both required for axon pruning, their exact order of activation during axon pruning in neurons is still unclear.