Figure 4.
Direct activation of necroptosis triggers SARM1 dependent axon degeneration.(A) Top: Schematic of MLKL and dimerizable MLKL.ND construct (MLKL.ND = MLKL N-terminal domain) used in this study. MLKL.ND is fused to FkbpF36V. Bottom: Neurons are infected with lentivirus expressing MLKL.ND, and dimerization is induced with BB (100 nM) to promote necroptosis. (B) Representative images of cell bodies of cultured DRG neurons expressing MLKL.ND at baseline (left) and 24 h after BB (100 nM) application (right). After 24 h, most cells have died from necroptosis (lose both Hoechst staining and soluble GFP fluorescence: gain both annexin V and EthD staining). In two cells (arrow), annexin V is detected before EthD staining or loss of both Hoechst and cytosolic GFP, suggesting that annexin V staining is an early sign of impending cell death. Hoechst (blue), cytosolic GFP (green), annexin V (red), EthD (yellow); scale bar, 30 µm. (C) Representative bright-field and TMRM images of axons from WT and SARM1 KO DRG neurons expressing MLKL.ND at baseline (left) and 24 h after BB (100 nM) treatment (right). Scale bar, 50 µm. (D) Quantification of axon degeneration (left, n = 5) and TMRM intensity (right, n = 3) at the indicated time points after BB (100 nM) addition to WT or SARM1 KO neurons. Axons with a DI >0.3 are defined as degenerated (dashed line). Data represent the mean ± SEM. For axon degeneration, two-way ANOVA with post hoc Bonferroni’s multiple comparison test; time, F(2,16) = 106.5, P < 0.0001; interaction, F(8,16) = 4.635, P = 0.0044. For TMRM, two-tailed unpaired t test, P < 0.0001; *, P < 0.05; **, P < 0.01; and ***, P < 0.001. (E) Representative fluorescent images of WT or SARM1 KO axons infected with lentivirus encoding the calcium reporter GCaMP6, the axon integrity reporter mRuby3, and MLKL.ND at baseline and 21 h after BB application. Scale bar, 30 µm. (F) Depiction of Ca2+ dynamics with GCaMP6 fluorescent intensity (green, left y axis), and axon integrity with mRuby3 fluorescent intensity (red, right y axis) from a representative single axon of each genotype from E (arrowhead).

Direct activation of necroptosis triggers SARM1 dependent axon degeneration. (A) Top: Schematic of MLKL and dimerizable MLKL.ND construct (MLKL.ND = MLKL N-terminal domain) used in this study. MLKL.ND is fused to FkbpF36V. Bottom: Neurons are infected with lentivirus expressing MLKL.ND, and dimerization is induced with BB (100 nM) to promote necroptosis. (B) Representative images of cell bodies of cultured DRG neurons expressing MLKL.ND at baseline (left) and 24 h after BB (100 nM) application (right). After 24 h, most cells have died from necroptosis (lose both Hoechst staining and soluble GFP fluorescence: gain both annexin V and EthD staining). In two cells (arrow), annexin V is detected before EthD staining or loss of both Hoechst and cytosolic GFP, suggesting that annexin V staining is an early sign of impending cell death. Hoechst (blue), cytosolic GFP (green), annexin V (red), EthD (yellow); scale bar, 30 µm. (C) Representative bright-field and TMRM images of axons from WT and SARM1 KO DRG neurons expressing MLKL.ND at baseline (left) and 24 h after BB (100 nM) treatment (right). Scale bar, 50 µm. (D) Quantification of axon degeneration (left, n = 5) and TMRM intensity (right, n = 3) at the indicated time points after BB (100 nM) addition to WT or SARM1 KO neurons. Axons with a DI >0.3 are defined as degenerated (dashed line). Data represent the mean ± SEM. For axon degeneration, two-way ANOVA with post hoc Bonferroni’s multiple comparison test; time, F(2,16) = 106.5, P < 0.0001; interaction, F(8,16) = 4.635, P = 0.0044. For TMRM, two-tailed unpaired t test, P < 0.0001; *, P < 0.05; **, P < 0.01; and ***, P < 0.001. (E) Representative fluorescent images of WT or SARM1 KO axons infected with lentivirus encoding the calcium reporter GCaMP6, the axon integrity reporter mRuby3, and MLKL.ND at baseline and 21 h after BB application. Scale bar, 30 µm. (F) Depiction of Ca2+ dynamics with GCaMP6 fluorescent intensity (green, left y axis), and axon integrity with mRuby3 fluorescent intensity (red, right y axis) from a representative single axon of each genotype from E (arrowhead).

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