Article Figures & Data
Figures
- Figure 1.
Upregulation of core necroptosis components in the spinal cord of symptomatic Tg SOD1G93A mice. Lumbar spinal cords, from 12- and 15-week-old Tg SOD1G93A, WT SOD1, and NTg mice, were isolated and processed for mRNA and protein (RIPA or urea extraction) expression of RIPK1, RIPK3, MLKL, and p-MLKL. A, Quantification of Ripk1, Ripk3, and Mlkl mRNA from 12-week-old mice. Gapdh: housekeeping gene. A significant increase was detected for Ripk3 in Tg SOD1G93A compared to Tg SOD1WT (p = 0.0021) and NTg (p = 0.0076) at 12 weeks. B, Quantification of Ripk1, Ripk3, and Mlkl mRNA in spinal cords of 15-week-old mice. Gapdh: housekeeping gene. A significant increase was detected for Ripk1 (p = 0.0009, vs Tg SOD1WT; p = 0.0020, vs NTg) and Ripk3 (p = 0.0115; vs Tg SOD1WT, p = 0.0067; vs NTg) but not for Mlkl in Tg SOD1G93A compared to Tg SOD1WT and NTg mice at 15 weeks. C, Western blotting (RIPA) for RIPK1 in spinal cord of NTg, Tg SOD1WT, and Tg SOD1G93A 15-week-old mice. β-ACTIN, GAPDH: loading control. Specificity of the RIPK1 band was confirmed following downregulation of RIPK1 with specific lentiviral shRNA in mouse PMN cultures (mPMNs). D, Quantification of RIPK1 protein levels. RIPK1 protein is significantly increased in Tg SOD1G93A samples compared to Tg SOD1WT (p = 0.0279) and NTg (p = 0.0033) mice. Results are presented as mean ± SEM. Statistical analysis was performed via one-way ANOVA followed by Tukey’s post hoc analysis; n = 3 biological replicates per genotype. E, Western blotting (RIPA) for RIPK3 showed no specific signal at the expected 55 kDa in spinal cord (NTg and Tg SOD1G93A). Non-specific band at 47 kDa is designated as an asterisk (*). NTg spleen: positive control tissue, Ripk3−/− spleen and Tg SOD1G93A;Ripk3−/− spinal cord: negative control tissue. F, Western blotting (urea) for RIPK3 antibody showed no specific signal at the expected 55 kDa in spinal cord (NTg and Tg SOD1G93A). Ripk3−/− spinal cord: negative control tissue. G, Western blotting (RIPA) for MLKL showed no specific signal at the expected 55 kDa in spinal cord. NIH 3T3: positive control cell lysate. H, Western blotting (urea) for MLKL showed no specific signal at the expected 55 kDa in spinal cord (NTg and Tg SOD1G93A). NIH 3T3: positive control cell lysate. I, Western blotting for p-MLKL (RIPA) showed no signal at the expected 55 kDa. TSZ-treated L929 cells: control cell lysate.
- Figure 2.
RIPK1 expression in brain cortex from ALS patients. Postmortem motor cortex (Brodmann’s area 4) from sporadic ALS, SOD1 ALS, and non-ALS human brains was homogenized and processed for RIPK1 protein expression. Western blotting for RIPK1 protein. Two different antibodies against RIPK1 were used (A, AB_397831; B, AB_394014). C, Quantification of RIPK1 protein levels. β-ACTIN, loading control. No significant differences were detected for RIPK1 between sporadic ALS, SOD1 ALS, and non-ALS human brain samples. Results are presented in a scatter dot plot. Line = mean. Statistical analysis was performed via Student’s t test: t(10) = 0.579, p = 0.575; n = 4; non-ALS, n = 6; sporadic ALS, and n = 2; SOD1 ALS.
- Figure 3.
Ripk3−/− MNs are resistant to Tg SOD1G93A astrocyte-mediated toxicity. MNs, isolated from E12.5 Ripk3+/+ or Ripk3−/− mice, were co-cultured on primary astrocyte monolayers from Tg SOD1G93A or NTg mice for 7 d. A, Representative images of MNs assessed using SMI32 immunolabeling. Scale bar: 50 μm. B, Quantification of MN number. Ripk3+/+ MN number was significantly reduced on SOD1G93A astrocytes (p = 0.0013) compared to NTg. Ripk3−/− MN number did not differ between NTg or SOD1G93A astrocytes and was significantly increased (p = 0.0370) compared to Ripk3+/+ MN number on SOD1G93A astrocytes. Results are presented as a mean ± SEM. Statistical analysis was performed via two-way ANOVA; n = 3 biological replicates per genotype. *p ≤ 0.05; **p ≤ 0.01.
- Figure 4.
Onset and survival in Tg SOD1G93A;Ripk3−/− mice. Mice were separated by genotype and sex for onset (%) and survival (%), and data were plotted as Kaplan–Meier curves. A, Onset in Tg SOD1G93A;Ripk3−/− (median = 155) and Tg SOD1G93A;Ripk3+/+ (median = 151), p = 0.06; male and female mice. B, Onset in Tg SOD1G93A;Ripk3−/− (median = 155) and Tg SOD1G93A;Ripk3+/+ (median = 152), p = 0.005; male mice. C, Onset in Tg SOD1G93A;Ripk3−/− (median = 148) and Tg SOD1G93A;Ripk3+/+ (median = 155), p = 0.836; female mice. D, Survival in Tg SOD1G93A;Ripk3−/− (median = 169) and Tg SOD1G93A;Ripk3+/+ (median = 163), p = 0.145; male and female mice. E, Survival in Tg SOD1G93A;Ripk3−/− (median = 178) and Tg SOD1G93A;Ripk3+/+ (median = 157), p = 0.017; male mice. F, Survival in Tg SOD1G93A;Ripk3−/− (median = 163) and Tg SOD1G93A;Ripk3+/+ (median = 170), p = 0.340; female mice. For A, D, 10 Tg SOD1G93A;Ripk3+/+ mice and 12 Tg SOD1G93A;Ripk3−/− mice. For B, E, 5 Tg SOD1G93A;Ripk3+/+ mice and 7 Tg SOD1G93A;Ripk3−/− mice. For C, F, 5 Tg SOD1G93A;Ripk3+/+ mice and 5 Tg SOD1G93A;Ripk3−/− mice. Statistical analysis was performed via log-rank Mantel–Cox test.
- Figure 5.
Functional motor tests on Tg SOD1G93A;Ripk3−/− mice. Mice were separated by genotype and sex for grip strength analysis. A, Inverted grid score in Tg SOD1G93A;Ripk3−/− and Tg SOD1G93A;Ripk3+/+ male and female mice. B, Inverted grid score in Tg SOD1G93A;Ripk3−/− and Tg SOD1G93A;Ripk3+/+ male mice. C, Inverted grid score in Tg SOD1G93A;Ripk3−/− and Tg SOD1G93A;Ripk3+/+ female mice. D, Loaded grid score in Tg SOD1G93A;Ripk3−/− and Tg SOD1G93A;Ripk3+/+ male and female. E, Loaded grid score in Tg SOD1G93A;Ripk3−/− and Tg SOD1G93A;Ripk3+/+ male mice. F, Loaded grid score in Tg SOD1G93A;Ripk3−/− and Tg SOD1G93A;Ripk3+/+ female mice. For A, D, 14 Tg SOD1G93A;Ripk3+/+ mice and 17 Tg SOD1G93A;Ripk3−/− mice. For B, E, 8 Tg SOD1G93A;Ripk3+/+ mice and 10 Tg SOD1G93A;Ripk3−/− mice. For C, F, 6 Tg SOD1G93A;Ripk3+/+ mice and 7 Tg SOD1G93A;Ripk3−/− mice. Statistical analysis was performed by unpaired t test, two-tailed in selected time points using post hoc Bonferroni correction.
- Figure 6.
MN number and NMJ innervation is not improved in Tg SOD1G93A;Ripk3−/− mice. A, Representative images of ChAT+ cells in lumbar (L4-L5) ventral horns from P140 (close to end-stage) mice (fluorescent microscope; 10× magnification). Scale bar: 50 μm. B, Quantification of MN number, expressed as average number of neurons per hemisection (20 μm). Statistical significant difference was detected between NTg (Ripk3+/+ and Ripk3 −/−) and Tg (SOD1G93A;Ripk3+/+ and SOD1G93A;Ripk3−/−), p < 0.0001. No difference was detected between Tg SOD1G93A;Ripk3−/− and Tg SOD1G93A;Ripk3+/+ mice. Results are presented as a mean ± SEM. Statistical analysis was performed via two-way ANOVA followed by Newman–Keuls post hoc test; n = 3 biological replicates per genotype. C, Representative images of NMJ assessed by the expression of BTX (red, postsynaptic) and VAChT (green, presynaptic) in the TA muscle of P120 mice. Left column, Tg SOD1G93A;Ripk3+/+. Right column, Tg SOD1G93A;Ripk3−/− mice. Scale bar: 100 μm. D, Quantification of NMJs from 20-μm sections (n = ∼100 NMJs). NMJs were categorized as innervated (complete colocalization of BTX and VAChT), partial (partial colocalization of BTX and VAChT), or denervated (no colocalization between BTX and VAChT) and are presented as a percentage of the total NMJ number counted. No difference was observed in the number of innervated, partially innervated, and denervated NMJs between Tg SOD1G93A;Ripk3−/− and Tg SOD1G93A;Ripk3+/+ mice. Results are presented as a mean ± SEM. Statistical analysis was performed via unpaired Student’s t test, two-tailed; n = 3 biological replicates per genotype. ***p ≤ 0.001.
- Figure 7.
MN myelination morphology is not impaired in Tg SOD1G93A P90 mice. A, Gross morphology of the spinal cords (L1-L5) in transverse sections. B, Enlarged boxed areas in A indicate areas of white matter of ventral horn with adjacent ventral root. Note myelin degeneration only in Tg SOD1G93A (arrows in B, marked only few). A, B, Semithin sections, stained with toluidine blue. C, Ultrastructure of myelinated fibers in white matter of ventral horn. D, Ultrastructure of ventral roots. Note that myelin morphology is not impaired in both genotypes in C, D. All images in the horizontal panels were obtained at the same magnification. E, Ultrastructure of white matter ventral horn. F, Enlarged boxed areas in E. Note the decompacted morphology of myelin in both genotypes. Vertical columns correspond to the mice genotype indicated at the top. Scale bars: 0.5 cm (A), 25 μm (B), 10 μm (C–E), 5 μm (F).
- Figure 8.
Characterization of the Optn−/− mouse. A, Schematic of the Optn−/− mice. Optn knock-out allele was generated by two steps crossing of FLPase and CRE mice separately with Optn tm1a(EUCOMM)Wtsi mice to trim out the critical exon. B, Western blotting for OPTN expression in Optn+/− (+/−), Optn−/− (−/−), and control (+/+) spinal cord tissue. GAPDH, loading control. C, Representative images of ChAT+ cells in lumbar ventral spinal cord (L4-L5) from one-year-old Optn+/+ and Optn−/− mice. Scale bar: 50 μm. D, Quantification of medial motor column (MMC) MN number, expressed in average number of neurons per hemisection. Results are presented as a mean ± SEM. Statistical analysis was performed via unpaired Student’s t test; n = 3 biological replicates per genotype. E, Representative images of NMJ, of the TA muscle, assessed by the expression of BTX (red, postsynaptic) and Syn (green, presynaptic) from one-year-old Optn−/− mice. Colocalization of BTX and Syn represents an innervated NMJ. Scale bar: 50 μm. F, Quantification of innervated NMJs (%) in the Optn+/+, Optn+/−, and Optn−/− mice. Results are presented as mean ± SEM. Statistical analysis was performed via unpaired Student’s t test, two-tailed; n = 3 biological replicates per genotype. G, Ultrastructure of white matter ventral horn in the Optn+/+ and Optn−/− mice. H, Ultrastructure of ventral roots in the Optn+/+ and Optn−/− mice. Note that myelin does not differ in both genotypes of mice in G, H. Scale bar: 10 μm (G, H).
Tables
Disease Sex Age at death PMI cold (HH:MM) PMI frozen (HH:MM) Age at onset Disease duration (months) Notes NON-ALS #1 M 82 4:55 11:20 . . Neuro normal NON-ALS #2 F 54 6:41 16:36 . . Neuro normal NON-ALS #3 F 62 4:33 4:12 . . Hypoxic ischemic encephalopathy NON-ALS #4 M 60 N/A 10:08 . . Neuro normal SALS #5 M 67 4:40 19:20 65 19 Right lower extremity onset SALS #6 M 56 4:53 10:57 54 18 Lower extremity onset SALS #7 F 72 3:01 19:57 70 29 Diffuse weakness, proximal involvement SALS #8 M 56 N/A 13:17 54 33 Lower extremity onset, used high dose steroids (prednisone, decadron, testosterone), angiogenin mutation (I70V) SALS #9 F 71 N/A 5:11 70 30 Bulbar onset SALS #10 F 69 5:30 5:00 67 18 Bulbar onset FALS #11 F 52 12:45 4:00 50 24 SOD1 mutation (L144F) FALS #12 F 32 N/A 5:10 29 30 SOD1 mutation (A5V) SALS, sporadic ALS; FALS; familial ALS; M, male; F, female, N/A, not available; PMI, postmortem interval.
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