Mitochondrial dysfunction induces Sarm1-dependent cell death in sensory neurons

Abstract

Mitochondrial dysfunction is the underlying cause of many neurological disorders, including peripheral neuropathies. Mitochondria rely on a proton gradient to generate ATP and interfering with electron transport chain function can lead to the deleterious accumulation of reactive oxygen species (ROS). Notably, loss of mitochondrial potential precedes cellular demise in several programmed cell destruction pathways, including axons undergoing Wallerian degeneration. Here, we demonstrate that mitochondrial depolarization triggers axon degeneration and cell death in primary mouse sensory neurons. These degenerative events are not blocked by inhibitors of canonical programmed cell death pathways such as apoptosis, necroptosis, and parthanatos. Instead, the axodestructive factor Sarm1 is required for this axon degeneration and cell death. In the absence of Sarm1, the mitochondrial poison CCCP still induces depolarization of mitochondria, ATP depletion, calcium influx, and the accumulation of ROS, yet cell death and axon degeneration are blocked. The survival of these neurons despite the accumulation of ROS indicates that Sarm1 acts downstream of ROS generation. Indeed, loss of Sarm1 protects sensory neurons and their axons from prolonged exposure to ROS. Therefore, Sarm1 functions downstream of ROS to induce neuronal cell death and axon degeneration during oxidative stress. These findings highlight the central role for Sarm1 in a novel form of programmed cell destruction that we term sarmoptosis.

Keywords: Sarm1; axon; cell death; degeneration; mitochondria; reactive oxygen species.

Figure 1.

Mitochondrial dysfunction stimulates Sarm1-dependent axon degeneration and cell death. A, Bright-field images from a field of neuronal distal axons treated with 50 μm CCCP for the indicated time. Right, Quantification of axon degeneration after CCCP treatment (n = 3). B, Bright-field images from a field of neuronal soma treated with 50 μm CCCP during the same time course experiment as in A. Neuronal soma are incubated with 200 nm ethidium homodimer (red). Arrowheads indicate examples of membrane protrusions from dying soma. Quantification of cell death is shown on the right (n = 3). C, D, Quantification of axon degeneration from WT and Sarm1^−/− neurons treated with 50 μm CCCP for 24 h or treated with 25 μm rotenone for 36 h (n = 3). Representative bright-field images of distal axons. E, F, Quantification of cell death from WT and Sarm1^−/− neurons treated as in C (n = 3). Shown are representative bright-field images of neuronal soma incubated with ethidium homodimer (red). Error bars represent SEM. Scale bars, 25 μm. *p < 0.05; **p < 0.01.

Figure 2.

Analysis of structural features required for Sarm1 action in CCCP-induced degeneration and death. A, Domain structure of Sarm1 with location of alanine replacement mutations highlighted. Sarm1^−/− neurons were transduced with lentiviruses expressing the indicated construct. At DIV7, neurons were treated with 50 μm CCCP and then axon degeneration (B) and cell death (C) were measured (n = 3). Error bars represent SEM. Statistically significant differences are noted in the figure. *p < 0.05; **p < 0.01.

Figure 3.

Mitochondrial dynamics of depolarized mitochondria in WT and Sarm1^−/− neurons. A, WT and Sarm1^−/− neurons were loaded with TMRM to label polarized mitochondria. Neurons were treated with DMSO or 50 μm CCCP and bright-field and TMRM fluorescent images were acquired. Scale bar, 25 μm. B, Lysates of WT and Sarm1^−/− neurons treated with DMSO or 50 μm CCCP for 4 h were analyzed by Western blotting with the indicated antibodies. C, Quantification of mitochondria from WT and Sarm1^−/− neurons treated with DMSO or 50 μm CCCP for 4 h (n = 3). D, Population analysis of mitochondrial shape in WT and Sarm1^−/− neurons. The ratio of major to minor axis of the mitochondria was used to calculate “roundness” and is displayed as a cumulative frequency distribution. Mitochondria were also measured in neurons treated with 50 μm CCCP for 4 h. E, Images of axonal mitochondria from WT and Sarm1^−/− neurons treated with 50 μm CCCP or DMSO for 4 h. Scale bar, 12 μm. F, Kymographs of mitochondrial movement in WT and Sarm1^−/− neurons treated with DMSO or 50 μm CCCP for 4 h. G, Quantification of percentage motile mitochondria from WT or Sarm1^−/− neurons treated with 50 μm CCCP or DMSO for 4 h (n = 3). Statistical analysis did not reveal any differences between WT and Sarm1^−/− neurons. Error bars represent SEM.

Figure 4.

Sarm1^−/− neurons use glycolysis to survive prolonged mitochondrial depolarization. A, Measurement of ATP levels as a percentage of control (DMSO) from WT and Sarm1^−/− neurons during CCCP treatment. B, l-Lactate levels in media were measured from WT and Sarm1^−/− neurons treated with DMSO or 50 μm CCCP (n = 4). Data are represented as a percentage of control. C, Axon degeneration in distal axons from WT and Sarm1^−/− treated with DMSO, 50 μm CCCP, 5 mm 2-DG, or 50 μm CCCP + 5 mm 2-DG (n = 3). Below are representative images from neurons treated as described. D, Cell death in WT and Sarm1^−/− neurons treated as in C (n = 3). Below are representative images of neuronal soma stained with ethidium homodimer (red). Axon degeneration and cell death were quantified 24 h after treatment. Scale bars, 25 μm. Statistically significant differences are noted in the figure. *p < 0.05; **p < 0.01.

Figure 5.

Analysis of programmed cell death pathways in CCCP-induced axon degeneration and cell death. WT neurons were transduced with lentivirus expressing BclxL or pretreated with the pan-caspase inhibitor Z-VAD-fmk (10 μg/ml). Neurons were then treated with 50 μm CCCP or deprived of NGF and analyzed 24 h later for axon degeneration (A) and cell death (B). In C and D, WT neurons were treated with actinomycin D (1 μg/ml) and with CCCP as described for A and B. Axon degeneration and cell death were measured 24 h later. In E and F, WT neurons were treated with indicated compound: ferrostatin 1 (Fer-1; 1 μm), necrostatin 1 (Nec-1; 5 μm), olaparib (1 μm), or rucaparib (0.5 μm). Neurons were then treated with 50 μm CCCP and analyzed 24 h later for axon degeneration and cell death (n = 3). G, H, Neurons were infected with DIO SAM-TIR lentivirus that expresses Sarm1 SAM-TIR domain in a Cre-dependent fashion. Two days later, the neurons were infected with Cre-expressing while in the presence of the indicated inhibitors. Thirty-six hours later, cells were assessed for axon degeneration (G) and cell death (H) (n = 3). Error bars represent SEM. Statistically significant differences are noted in the figure. **p < 0.01; ***p < 0.001.

Figure 6.

Calcium influx during mitochondrial dysfunction. Neurons were treated with 50 μm CCCP alone or in presence of ALLN (25 μm) or EGTA (2.5 mm). A, B, Axon degeneration (A) or cell death (B) were measured 24 h after treatment (axon degeneration: *p = 0.001; cell death: *p = 0.01; two-sample t test). C, Images of WT and Sarm1^−/− neuronal soma were preincubated with the calcium indicator dye Fluo-4AM. Neurons were treated with DMSO or 50 μm CCCP for 8 h. Quantification of Fluo-4AM intensity of the soma is shown on the right (n = 3). D, Images of Fluo-4AM fluorescence in distal axons from WT and Sarm1^−/− neurons (n = 3) treated as in C. E, Axons from WT and Sarm1^−/− DRGs were transected and Fluo4AM intensity measured in distal axons at the indicated times after axotomy (n = 4). Quantification of the ratio of Fluo-4AM intensity to total axon area (per 500 pixels) is shown on the right. Fluo4-AM intensity was not statistically different between WT and Sarm1^−/− neurons in any of the experiments. Scale bars, 25 μm.

Figure 7.

Mitochondrial dysfunction induces ROS generation in WT and Sarm1^−/− neurons. A, WT and Sarm1^−/− neurons were treated with DMSO or 50 μm CCCP for 8 h. Neurons were loaded with the ROS indicator dye DHE and images of DHE fluorescence were acquired. Bright-field images of soma were also acquired and merged images are shown at right. Scale bar, 25 μm. B, Quantification of DHE fluorescence in neurons treated as in A (n = 3). Statistical analysis indicates no significant difference in DHE intensity after CCCP treatment between WT and Sarm1^−/− DRGs. C, Immunofluorescence images of WT and Sarm1^−/− neurons treated as in A and stained for HNE, a product of lipid peroxidation. Note the equivalent staining in both WT and Sarm1^−/− neurons. Scale bar, 20 μm. Error bars represent SEM.

Figure 8.

Sarm1^−/− neurons are resistant to oxidative stress. A, WT and Sarm1^−/− neurons were treated with the indicated doses of hydrogen peroxide (H₂O₂). Axon degeneration was measured 24 h after treatment (n = 3). B, Neurons were treated as in A and cell death was measured 24 h after treatment (n = 3). Below are representative images of distal axons and soma (treated with EH to label dead cells). C, D, Quantification of axon degeneration (C) and cell death (D) in neurons treated with 25 μm paraquat for 24 h (n = 3). Error bars represent SEM. Scale bars, 25 μm. Statistically significant differences are noted in the figure. *p < 0.05; **p < 0.01.