Fig 1: rADAMTS-4 accelerates neurodegeneration in the lumbar spinal cord of SOD1G93A mice. a Representative photomicrographs of ventral horns in lumbar spinal cord sections from WT and control or ADAMTS-4-treated SOD1G93A male mice stained with ChAT. Scale bar: 500 or 250 μm. b-c Quantification of average spinal motoneuron number (b) and size (c) in male mice from (a). Values plotted are mean ± SEM. Two-way ANOVA: *** P < 0.001 WT Vs SOD1G93A. Unpaired two-tailed t-Test: P > 0.05 (Number) or $ P < 0.05 (Size) Control Vs ADAMTS-4 SOD1G93A, N = 3 Control WT, N = 5 ADAMTS-4 WT, N = 8 Control SOD1G93A, N = 7 ADAMTS-4 SOD1G93A. d Representative photomicrographs of ventral horns in lumbar spinal cord sections from WT and control or ADAMTS-4-treated SOD1G93A female mice stained with ChAT. Scale bar: 500 or 250 μm. e-f Quantification of average spinal motoneuron number (e) and size (f) in female mice from (d). Values plotted are mean ± SEM. Two-way ANOVA: *** P < 0.001 WT Vs SOD1G93A. Unpaired two-tailed t-Test: $ P < 0.05 (Number) or P > 0.05 (Size) Control Vs ADAMTS-4 SOD1G93A, N = 5 Control WT, N = 3 ADAMTS-4 WT, N = 5 Control SOD1G93A, N = 6 ADAMTS-4 SOD1G93A
Fig 2: rADAMTS-4 reduces perineuronal nets enwrapping motoneurons in the lumbar spinal cord of SOD1G93A mice. a Representative photomicrographs of ventral horns in lumbar spinal cord sections from WT and control or ADAMTS-4-treated SOD1G93A male mice stained with WFA, a marker of perineuronal nets. Scale bar: 500 or 250 μm. b Quantification of WFA immunoreactivity per area from male mice (a). Values plotted are mean ± SEM. Two-way ANOVA: *** P < 0.001 WT Vs SOD1G93A. Unpaired two-tailed t-Test: $$ P < 0.01 Control Vs ADAMTS-4 SOD1G93A, N = 3 Control WT, N = 5 ADAMTS-4 WT, N = 8 Control SOD1G93A, N = 7 ADAMTS-4 SOD1G93A. c Representative photomicrographs of ventral horns in lumbar spinal cord sections from WT and control or ADAMTS-4-treated SOD1G93A female mice stained with WFA. Scale bar: 500 or 250 μm. d Quantification of WFA immunoreactivity per area from female mice (c). Values plotted are mean ± SEM. Two-way ANOVA: *** P < 0.001 WT Vs SOD1G93A. Unpaired two-tailed t-Test: $ P < 0.05 Control Vs ADAMTS-4 SOD1G93A, N = 5 Control WT, N = 3 ADAMTS-4 WT, N = 5 Control SOD1G93A, N = 6 ADAMTS-4 SOD1G93A. e Positive correlation between the percentage of WFA-positive perineuronal nets per area and the number of motoneurons in male and female WT (N = 15; blank diamond) and SOD1G93A (N = 24; black diamond) symptomatic mice from figs. 4–5. Spearman’s rank correlation: *** P < 0.001. R represents the coefficient of correlation. f-k Quantitative RT-PCR for aggrecan (f-g) HAPLN1 (h-i) and tenascin R (j-k) in the lumbar spinal cord of WT and SOD1G93A male (f, h, j) and female (g, i, k) mice at PS, SS and ES. Values plotted are mean ± SEM. Mann–Whitney U-tests: * P < 0.05 compared to corresponding WT mice, $ P < 0.05 compared to SOD1G93A mice at other stages, # P < 0.05 compared to WT mice at other stages, N = 3-4. l Immunoblot for CSPG (chondroitin sulfate proteoglycans) core proteins in lumbar spinal cord protein extracts of SOD1G93A mice exposed to human recombinant ADAMTS-1, ADAMTS-4 or ADAMTS-5 ex vivo for 24 h at 37 °C. Quantification of total CSPG core proteins. Values plotted are mean ± SEM. Mann–Whitney U-tests: * P < 0.05 between control and ADAMTS-4 conditions, N = 4
Fig 3: ADAMTS-4 modulates the synthesis/release of neurotrophic factors by glial cells in vitro. a-c Quantitative RT-PCR for NGF (a) GDNF (b) and BDNF (c) expression in mouse adult cortical astrocyte cultures treated or not for 48 h with a human recombinant ADAMTS-4 (20, 100, 200 ng/ml). Values plotted are mean ± SEM. Mann–Whitney U-tests: * P < 0.05 compared to control, N = 4. d-e ELISA-measurements of NGF released in the media of mouse adult cortical astrocyte cultures treated or not for 48 h with a human recombinant ADAMTS-4 (d) or ADAMTS-1 (e) at different doses (20, 100, 200 ng/ml). Values plotted are mean ± SEM. Mann–Whitney U-tests: * P < 0.05 (ADAMTS-4) or P > 0.05 (ADAMTS-1) compared to control, N = 3. f-i Quantitative RT-PCR for ADAMTS-4 (f) NGF (g) GDNF (h) and BDNF (i) expression in mouse adult cortical astrocyte cultures transfected or not for 48 h with empty vector (mock) or silencing RNAs (siRNAs) targeting the expression of ADAMTS-4. Values plotted are mean ± SEM. Mann–Whitney U-tests: * P < 0.05 (ADAMTS-4, NGF, GDNF) or P > 0.05 (BDNF) compared to mock, N = 4. j ELISA-measurements of NGF released in the media of mouse adult cortical astrocyte cultures transfected or not for 48 h with mock or siRNAs targeting the expression of ADAMTS-4. Values plotted are mean ± SEM. Mann–Whitney U-tests: * P < 0.05 compared to control, N = 4. k-m Quantitative RT-PCR for NGF (k) GDNF (l) and BDNF (m) expression in mouse neonatal cerebral microglia cultures treated or not for 48 h with a human recombinant ADAMTS-4 (20, 100, 200 ng/ml). Values plotted are mean ± SEM. Mann–Whitney U-tests: * P < 0.05 compared to control, N = 4. n-q Quantitative RT-PCR for ADAMTS-4 (n) NGF (o) GDNF (p) and BDNF (q) expression in mouse neonatal cerebral microglia cultures transfected or not for 48 h with mock or siRNAs targeting the expression of ADAMTS-4. Values plotted are mean ± SEM. Mann–Whitney U-tests: * P < 0.05 (NGF, BDNF), *** P < 0.001 (ADAMTS-4, GDNF) compared to mock, N = 8
Fig 4: Gender similarities and differences in the effect of ADAMTS-4 treatment on ALS. a Schematic representation of ADAMTS-4 treatment promoting the decline of NGF production and ALS-induced perineuronal net degradation which contribute to the degeneration and even death of motoneurons in the ventral horn of the lumbar spinal cord of SOD1G93A mice. b A table describing the similarities and differences observed in behavioral and anatomical effects of ADAMTS-4 treatment in SOD1G93A male and female mice
Fig 5: Presymptomatic treatment with rADAMTS-4 worsens the prognosis of SOD1G93A mice. a Kaplan-Meier graph showing the probability of symptom onset in SOD1G93A males treated with saline (Control; black line) or recombinant ADAMTS-4 (ADAMTS-4; gray line) at early presymptomatic stage. Log-rank (Mantel-Cox) Test: * P < 0.05 compared to the control group, N = 9. b Mean age at onset of mice from panel a. Values plotted are mean ± SEM. Mann–Whitney U-test: * P < 0.05 compared to the control group, N = 9. c The best wire hang performance during the first week after symptom onset. Values plotted are mean ± SEM. Mann–Whitney U-test: P > 0.05 compared to the control group, N = 9. d Kaplan-Meier graph showing the probability of symptom onset in SOD1G93A females treated with saline (Control; black line) or recombinant ADAMTS-4 (ADAMTS-4; gray line) during early presymptomatic stage. Log-rank (Mantel-Cox) Test: P > 0.05 compared to the control group, N = 7 Control SOD1G93A, N = 6 ADAMTS-4 SOD1G93A. e Mean age at onset of mice from panel D. Values plotted are mean ± SEM. Mann–Whitney U-test: P > 0.05 compared to the control group, N = 7 Control SOD1G93A, N = 6 ADAMTS-4 SOD1G93A. f The best wire hang performance during the first week after symptom onset. Values plotted are mean ± SEM. Mann–Whitney U-test: * P < 0.05 compared to the control group, N = 7 Control SOD1G93A, N = 6 ADAMTS-4 SOD1G93A. g-h Weight over time of WT and SOD1G93A male (g) or female (h) mice treated or not with saline or recombinant ADAMTS-4 during early presymptomatic stage. Values plotted are mean ± SEM. Two-way Anova: ** P < 0.01, *** P < 0.001 WT Vs SOD1G93A, N = 5 Control WT males, N = 5 ADAMTS-4 WT males, N = 9 Control SOD1G93A males, N = 9 ADAMTS-4 SOD1G93A males, N = 5 Control WT females, N = 4 ADAMTS-4 WT females, N = 7 Control SOD1G93A females, N = 6 ADAMTS-4 SOD1G93A females
Supplier Page from R&D Systems, a Bio-Techne Brand for Recombinant Human ADAMTS4 Protein, CF