Axonal Degeneration Is Mediated by Necroptosis Activation

Abstract

Axonal degeneration, which contributes to functional impairment in several disorders of the nervous system, is an important target for neuroprotection. Several individual factors and subcellular events have been implicated in axonal degeneration, but researchers have so far been unable to identify an integrative signaling pathway activating this self-destructive process. Through pharmacological and genetic approaches, we tested whether necroptosis, a regulated cell-death mechanism implicated in the pathogenesis of several neurodegenerative diseases, is involved in axonal degeneration. Pharmacological inhibition of the necroptotic kinase RIPK1 using necrostatin-1 strongly delayed axonal degeneration in the peripheral nervous system and CNS of wild-type mice of either sex and protected in vitro sensory axons from degeneration after mechanical and toxic insults. These effects were also observed after genetic knock-down of RIPK3, a second key regulator of necroptosis, and the downstream effector MLKL (Mixed Lineage Kinase Domain-Like). RIPK1 inhibition prevented mitochondrial fragmentation in vitro and in vivo, a typical feature of necrotic death, and inhibition of mitochondrial fission by Mdivi also resulted in reduced axonal loss in damaged nerves. Furthermore, electrophysiological analysis demonstrated that inhibition of necroptosis delays not only the morphological degeneration of axons, but also the loss of their electrophysiological function after nerve injury. Activation of the necroptotic pathway early during injury-induced axonal degeneration was made evident by increased phosphorylation of the downstream effector MLKL. Our results demonstrate that axonal degeneration proceeds by necroptosis, thus defining a novel mechanistic framework in the axonal degenerative cascade for therapeutic interventions in a wide variety of conditions that lead to neuronal loss and functional impairment.SIGNIFICANCE STATEMENT We show that axonal degeneration triggered by diverse stimuli is mediated by the activation of the necroptotic programmed cell-death program by a cell-autonomous mechanism. This work represents a critical advance for the field since it identifies a defined degenerative pathway involved in axonal degeneration in both the peripheral nervous system and the CNS, a process that has been proposed as an early event in several neurodegenerative conditions and a major contributor to neuronal death. The identification of necroptosis as a key mechanism for axonal degeneration is an important step toward the development of novel therapeutic strategies for nervous-system disorders, particularly those related to chemotherapy-induced peripheral neuropathies or CNS diseases in which axonal degeneration is a common factor.

Keywords: MLKL; RIP kinase; axonal degeneration; mitochondrial fragmentation; necroptosis; neurodegeneration.

Figure 1.

Pharmacological inhibition of RIPK1 delays degeneration of both peripheral and central axons. The degree of axonal degeneration was quantitatively assessed by immunofluorescence using NFH immunostaining in transverse nerve sections. A–D, Sciatic and optic nerve explants were treated with Nec-1 (100 μm) or vehicle (DMSO) for 48 h and 4 d, respectively. E, Axonal degeneration was evaluated in vivo in crushed sciatic nerves injected with 200 μl of 0.64 mm Nec-1 or vehicle (3% DMSO in PBS) 48 h after injury. Nec-1 treatments strongly delay axonal degeneration triggered by nerve injury. Scale bar, 20 μm. B, D, F, Quantification of NFH-positive axons in nerve cross sections was expressed as axonal density for both ex vivo explants (B, D) and in vivo crushed nerve (F). In injured nerves, statistically significant protection from axonal degeneration was seen after Nec-1 treatment compared with vehicle-treated nerves (^#p < 0.0001) or noninjured controls (*p < 0.0001). Mean values are shown, error bars indicate SEM, N = 3 per group; analysis was performed by one-way ANOVA test.

Figure 2.

Nerve function is protected by RIPK1 inhibition. The size of the CAP was measured in sciatic nerve explants cultured for 24 h in the presence or absence of Nec-1 (100 μm). A, CAP A-wave length and amplitude were registered in vehicle-treated nerves. B, The maximum peak of the CAP was analyzed. C, The recruitment of excitable fibers within the nerve was measured at fixed stimulus intensity and the maximum fast monophasic A-wave was obtained by increasing the intensity of the pulse. Nerves cultured for 24 h with Nec-1 showed a significantly higher response than nerves cultured under vehicle conditions. One-way ANOVA with post hoc Tukey; # indicates statistically significant compared with Veh; * indicates statistically significant compared with Ctrl.

Figure 3.

Mitochondrial fragmentation is associated with axonal degeneration after nerve injury. Mitochondrial morphology was evaluated along with axonal degeneration in teased fibers of sciatic nerve explants at 0, 24, and 48 h after injury. A, B, Axonal degeneration progression is shown in nerve transverse sections immunostained for neurofilament (A) and quantified as axonal density from NFH-positive axons per area (B). Scale bar, 20 μm. Mitochondria were immunodetected with a COX IV antibody (mitochondria, red) in teased sciatic nerves dissected from Thy1-YFP mice (axons, green). Morphological analysis of mitochondria was performed from COX IV+ labeling of binarized images (A, bottom). Scale bar, 5 μm. C, D, Mitochondrial length was plotted as relative frequency (C) and as mean values (D). E, Decreased mitochondrial length along axonal degeneration was associated with increased mitochondrial density, measured as number of mitochondria per area. F, Longitudinal sections of sciatic nerves were analyzed by electron microscopy, showing mitochondrial fragmentation and swelling at 24 and 48 h after injury, respectively. Scale bar, 1 μm. One-way ANOVA with post hoc Tukey, * indicates statistically significant compared with Ctrl.

Figure 4.

RIPK1 inhibition prevents axonal degeneration-associated mitochondrial fragmentation. A, Mitochondrial fragmentation was evaluated at 24 h after injury from binarized images of COX IV-immunostained teased nerves. B, Mitochondrion shortening was inhibited in nerves treated with Nec-1 (100 μm) or Mdivi (200 μm) compared with nerve explants treated with vehicle. C, Increased mitochondrial density was also inhibited by both treatments. D, E, Axonal integrity after Mdivi treatment was also evaluated by NFH staining of transversal nerve sections at 24 and 48 h after injury. One-way ANOVA with post hoc Tukey, * indicates statistically significant compared with Ctrl; # indicates statistically significant compared with Veh.

Figure 5.

RIPK1 activation and mitochondrial fragmentation contributes to axonal degeneration by a cell-autonomous mechanism. DRG explants were damaged by axotomy or vinblastine (1 μm) treatment in the presence of Nec-1, Mdivi, or vehicle as a control, and analyzed after 12 h. A, B, Degeneration was evaluated in distal axons immunostained for NFH (A), quantitatively assessed, and represented as relative neurite integrity (B). Scale bar, 30 μm. Two-way ANOVA with post hoc Tukey, * indicates statistically significant compared with Ctrl; # indicates statistically significant compared with Veh.

Figure 6.

Necroptosis is activated after axonal injury. Necroptotic pathway activation was evaluated after TNFα/z-VAD treatment through the detection of RIPK3, MLKL, and their phosphorylated forms by Western blot. A, Total protein (Coomassie staining) was used as a loading control to normalize the expression of the different proteins, which were expressed as fold induction compared with control (n = 3). Downstream activation of the pathway was determined in mechanically injured axons in a time course (≤6 h) through the detection of MLKL and p-MLKL. Results are represented as pMLKL/MLKL ratio from n = 5 independent experiments. Total protein was used as a loading control. Two-way ANOVA with post hoc Tukey, * indicates statistically significant compared with Ctrl; # indicates statistically significant compared with 3 h+Veh. B, C, Specificity of p-MLKL antibody was tested in injured axons devoid of MLKL by using DRGs transduced with lentiviral vectors containing MLKL shRNA (B) and compared with a control nontargeting shRNA (scramble; n = 3; C). D, The magnitude of MLKL or RIPK3 knock-down was evaluated by real-time qPCR in dissociated DRGs transduced with lentivirus containing shMLKL or shRIPK3, respectively (n = 3). One-way ANOVA with post hoc Tukey, * indicates significant compared with Scramble shRNA.

Figure 7.

Knock down of RIPK3 and MLKL in sensory neurons protects them from axonal degeneration in vitro. DRGs were transduced with lentiviral vectors containing RIPK3 or MLKL shRNA versus a control nontargeting shRNA (scramble). All plasmids express GFP for identification of transduced neurons. A, Representative images before and after injury (axotomy or vinblastine) are shown (NFH in red, GFP signal in green). Scale bar, 5 μm. B, C, Quantifications of axonal degeneration 8 h after axotomy (B) or 16 h after vinblastine (1 μm) treatment (C). Protection with shRIPK3 or shMLKL is represented with blue bars under both conditions. Two-way ANOVA with post hoc Tukey, * indicates statistically significant compared with Veh; # indicates statistically significant protection compared with axotomized or vinblastine-treated axons.