Death receptor 6 does not regulate axon degeneration and Schwann cell injury responses during Wallerian degeneration
- Department of Neurology, The Neuroscience Research Institute, College of Medicine, The Ohio State University Wexner Medical Center, United States
Figures
Figure 1 with 2 supplements
Rapid Wallerian degeneration in mice lacking DR6 in comparison to multiple mutants with delayed degeneration, assessed at 3 days after axotomy.
(A) Representative semithin (first and second columns) and electron micrographs (third column) of transverse sciatic nerve sections of distal nerve stumps from control and the indicated mutant mice 3 days after sciatic nerve transection. Note the complete structural disintegration of transected axons with absent or floccular cytoskeleton and collapsed myelin sheaths in the preparations from control, Tnfrsf21ΔEx2-3/ΔEx2-3, and Tnfrsf21ΔEx2/ΔEx2 mice. In contrast, the majority of disconnected axons from heterozygous WldS mice, and from Phr1 and SARM1 knockout mice, are structurally preserved with uniform cytoskeleton and intact myelin sheaths. Scale bars: 10 µm. (B) Quantification of preserved axons in transverse sciatic nerve sections of distal nerve stumps from mice with the indicated genotypes. Each symbol in the scatter dot plots represents the quantification from one animal (% of control axon numbers in micrographs from uninjured contralateral nerve preparations for each animal). All data were obtained from experimental animals between 3 and 12 months of age.
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Figure 1—source data 1
Numerical source data (axon survival quantification) for graphs shown in Figure 1B.
- https://cdn.elifesciences.org/articles/108389/elife-108389-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Normal rates of nerve injury-induced axon disintegration in DR6-deficient mice at earlier stages of Wallerian degeneration.
(A) Representative semithin micrographs of transverse tibial nerve sections of distal nerve stumps from 3-month-old mice with the indicated genotypes (top) 30 hr after sciatic nerve transection with pseudocoloring of intact (turquoise) and structurally degenerated (magenta) myelinated fibers, and corresponding quantifications of relative axon survival (bottom). (B) Representative semithin micrographs of transverse tibial nerve sections of distal nerve stumps from 3-month-old mice with the indicated genotypes (top) 36 hr after sciatic nerve transection with pseudocoloring of intact (turquoise) and structurally degenerated (magenta) myelinated fibers, and corresponding quantifications of relative axon survival (bottom). Scale bars: 10 µm.
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Figure 1—figure supplement 1—source data 1
Numerical source data (relative axon survival quantification) for graphs shown in Figure 1—figure supplement 1A and B.
- https://cdn.elifesciences.org/articles/108389/elife-108389-fig1-figsupp1-data1-v1.xlsx
Figure 1—figure supplement 2
Normal JNK activation in sciatic nerves from DR6-deficient mice.
(A, C) Representative western blots (cropped blot images) of lysates from uninjured (0 min) and axotomized distal sciatic nerve stumps (30 min after axotomy) from 1- to 2-month-old control and mutant mice with the indicated genotypes probed with the shown antibodies. Each lane represents the result from one mouse sciatic nerve. (B, D) Densitometric quantifications with statistical analysis of western blot data for all experimental animals used. Each dot represents a measurement from one sciatic nerve lysate from one mouse.
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Figure 1—figure supplement 2—source data 1
Numerical source data (densitometric p-JNK/JNK ratio quantification) for graphs shown in Figure 1—figure supplement 2B and D.
- https://cdn.elifesciences.org/articles/108389/elife-108389-fig1-figsupp2-data1-v1.xlsx
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Figure 1—figure supplement 2—source data 2
TIF file with original western blots and boxes indicating the relevant bands shown in Figure 1—figure supplement 2A and C.
- https://cdn.elifesciences.org/articles/108389/elife-108389-fig1-figsupp2-data2-v1.zip
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Figure 1—figure supplement 2—source data 3
Original files for western blot analysis displayed in Figure 1—figure supplement 2A and C.
- https://cdn.elifesciences.org/articles/108389/elife-108389-fig1-figsupp2-data3-v1.zip
Figure 2 with 1 supplement
Normal Schwann cell c-Jun injury responses during Wallerian degeneration (WD) in mice lacking DR6.
(A, C) Representative immunofluorescence confocal micrographs for c-Jun with DAPI nuclear counterstaining on transverse frozen sections from contralateral uninjured nerve (uncut) and distal sciatic nerve stumps 3 days and 30 hr following axotomy in 3-months-old mice with the indicated genotypes. (B, D) Corresponding quantifications of percentage of c-Jun immunoreactive and DAPI+ cells on nerve sections. Scale bars: 50 µm.
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Figure 2—source data 1
Numerical source data (cell counts) for graphs shown in Figure 2B and D.
- https://cdn.elifesciences.org/articles/108389/elife-108389-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Normal ultrastructure of transdifferentiated Schwann cells and normal myelin remodeling dynamics during Wallerian degeneration in DR6-deficient mice.
(A) Representative electron micrographs of transdifferentiated Schwann cells (red arrowheads) that lost axonal contact from distal sciatic nerve stumps 3 days following nerve transection in 3-month-old control and DR6 knockout mice with the shown genotypes. Note the similar ultrastructure with marked cytoplasmic expansion, increased organelle content, and myelin debris (myelin ovoids) in Schwann cell bodies in all genotypes. Scale bar: 2 µm. (B, C) Quantification of area occupied by myelin sheaths and myelin debris in transverse nerve sections from 3-month-old control and mutant mice with the indicated genotypes. Each dot represents the quantification obtained from one nerve obtained from one mouse.
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Figure 2—figure supplement 1—source data 1
Numerical source data (myelin area quantification) for graphs shown in Figure 2—figure supplement 1B and C.
- https://cdn.elifesciences.org/articles/108389/elife-108389-fig2-figsupp1-data1-v1.xlsx
Figure 3
Normal degeneration kinetics of injured dorsal root ganglion (DRG) neurites in primary neuronal cultures from DR6 knockout mice.
(A, D) Western blot analysis (cropped blot images) of DR6 protein expression in dorsal root ganglia (DRGs) from Tnfrsf21ΔEx2-3/ΔEx2-3 embryos and from Tnfrsf21Ex2LoxP/Ex2LoxP embryos infected with a lentivirus expressing Cre recombinase (DR6Cre), together with the respective control preparations. (B, E) Time course of neurite fragmentation, quantified as degeneration index (DI) (see Materials and methods) in DRG preparations from embryos with the indicated genotypes or lentiviral infection conditions. The data points shown in (B) represent the averaged neurite DI values calculated from multiple DRG preparations from each embryo at the indicated time point after injury (one data point = one embryo). The data points shown in (E) represent the averaged neurite DI values calculated from multiple micrographs acquired from each DRG preparation in a cell culture well at the indicated time point after injury (one data point = one well). (C, F) Representative phase-contrast micrographs of disconnected DRG neurites from embryos with the indicated genotypes and lentiviral infection conditions at the indicated time points after axotomy. Scale bars: 50 µm.
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Figure 3—source data 1
Numerical source data (degeneration index) for graphs shown in Figure 3B and E.
- https://cdn.elifesciences.org/articles/108389/elife-108389-fig3-data1-v1.xlsx
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Figure 3—source data 2
TIF file with original western blots and boxes indicating the relevant bands shown in Figure 3A and D.
- https://cdn.elifesciences.org/articles/108389/elife-108389-fig3-data2-v1.zip
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Figure 3—source data 3
Original files for western blot analysis displayed in Figure 3A and D.
- https://cdn.elifesciences.org/articles/108389/elife-108389-fig3-data3-v1.zip
Appendix 1—figure 1
Disrupted DR6 expression in two DR6 knockout mouse models.
(A, C) Quantitative real-time PCR analysis of relative brain Tnfrsf21 mRNA levels from control and mutant mice with the indicated genotypes. Each dot represents the measurement from one mouse. (B, D) Western blot analysis (cropped blot images) of brain lysates from control and mutant mice with the indicated genotypes probed with the shown antibodies. Each western blot lane represents the brain lysate data from one individual mouse.
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Appendix 1—figure 1—source data 1
Numerical source data (relative mRNA expression) for graphs shown in Appendix 1—figure 1A and C.
- https://cdn.elifesciences.org/articles/108389/elife-108389-app1-fig1-data1-v1.xlsx
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Appendix 1—figure 1—source data 2
TIF file with original western blots and boxes indicating the relevant bands shown in Appendix 1—figure 1B and D.
- https://cdn.elifesciences.org/articles/108389/elife-108389-app1-fig1-data2-v1.zip
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Appendix 1—figure 1—source data 3
Original files for western blot analysis displayed in Appendix 1—figure 1B and D.
- https://cdn.elifesciences.org/articles/108389/elife-108389-app1-fig1-data3-v1.zip
Appendix 1—figure 2
Normal developmental myelination in sciatic nerves from mice lacking DR6 at postnatal day 1.
(A, C) Representative semithin micrographs of transverse sciatic nerve sections from control and mutant mouse pups at postnatal day 1 with the indicated genotypes. Red arrows depict examples of axons with nascent myelination. Scale bars: 10 µm. (B, D) Quantification of myelinated axons in transverse sciatic nerve sections from control and mutant pups at postnatal day 1 with the indicated genotypes. Each dot represents the quantification obtained from one mouse pup.
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Appendix 1—figure 2—source data 1
Numerical source data (myelinated axon counts) for graphs shown in Appendix 1—figure 2B and D.
- https://cdn.elifesciences.org/articles/108389/elife-108389-app1-fig2-data1-v1.xlsx
Appendix 1—figure 3
Normal nerve histomorphometry in DR6-deficient mice.
(A, C) Quantification of g-ratios (left: scatter plots show g-ratios of individual myelinated axons as a function of axon diameter, right: corresponding cumulative g-ratios per animal) in tibial (A) and sciatic nerves (C) from 3-month-old control and mutant mice with the indicated genotypes. (B, D) Axon caliber and fiber caliber (axon plus myelin sheath) distributions in tibial (B) and sciatic nerves (D) from 3-month-old control and mutant mice with the indicated genotypes. Each dot represents the measurement from one mouse.
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Appendix 1—figure 3—source data 1
Numerical source data (histomorphometry quantification) for graphs shown in Appendix 1—figure 3.
- https://cdn.elifesciences.org/articles/108389/elife-108389-app1-fig3-data1-v1.xlsx
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/108389/elife-108389-mdarchecklist1-v1.docx
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Appendix 1—figure 1—source data 1
Numerical source data (relative mRNA expression) for graphs shown in Appendix 1—figure 1A and C.
- https://cdn.elifesciences.org/articles/108389/elife-108389-app1-fig1-data1-v1.xlsx
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Appendix 1—figure 1—source data 2
TIF file with original western blots and boxes indicating the relevant bands shown in Appendix 1—figure 1B and D.
- https://cdn.elifesciences.org/articles/108389/elife-108389-app1-fig1-data2-v1.zip
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Appendix 1—figure 1—source data 3
Original files for western blot analysis displayed in Appendix 1—figure 1B and D.
- https://cdn.elifesciences.org/articles/108389/elife-108389-app1-fig1-data3-v1.zip
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Appendix 1—figure 2—source data 1
Numerical source data (myelinated axon counts) for graphs shown in Appendix 1—figure 2B and D.
- https://cdn.elifesciences.org/articles/108389/elife-108389-app1-fig2-data1-v1.xlsx
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Appendix 1—figure 3—source data 1
Numerical source data (histomorphometry quantification) for graphs shown in Appendix 1—figure 3.
- https://cdn.elifesciences.org/articles/108389/elife-108389-app1-fig3-data1-v1.xlsx
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Death receptor 6 does not regulate axon degeneration and Schwann cell injury responses during Wallerian degeneration
eLife 14:RP108389.
https://doi.org/10.7554/eLife.108389.3
