Fig 1: 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 2: No modification of ADAMTS-5 expression in the lumbar spinal cord of SOD1G93A mice at any stage of the disease. a-f Immunoblot for ADAMTS-5 (73 kDa) in the lumbar spinal cord of WT and SOD1G93A male (a-c) and female (d-f) mice at PS, SS and ES. Values plotted are mean ± SEM. Mann–Whitney U-tests: P > 0.05 compared to corresponding WT mice, N = 4
Fig 3: ADAMTS-4 expression in the central nervous system. a Differential mRNA expression of ADAMTS proteoglycanases (eg. ADAMTS-1, −4, −5 and −9) in the lumbar spinal cord (SC) and in the cortex of 3-month-old WT male (♂) and female (♀) mice. Ct values are indicated in the histograms. Values plotted are mean ± SEM. Mann–Whitney U-tests: * P < 0.05 compared to ADAMTS-1 expression, $ P < 0.05 compared to ADAMTS-4 expression, # P < 0.05 compared to ADAMTS-5 expression, N = 3-4. b-d Representative photomicrographs of lumbar spinal cord sections from WT mice stained with: ADAMTS-4 (green) and NeuN (neuronal marker, red) (b) ADAMTS-4 (green) and GFAP (astrocyte marker, red) (c) or ADAMTS-4 (green) and APC (oligodendrocyte marker, red) (d). Corresponding Alexa fluor-488 negative controls for ADAMTS-4 (green) in the grey and white matter are shown in e
Fig 4: Reverse degradomics identification of ADAMTS5 substrates. A. Volcano plot showing significant ADAMTS5 cleaved peptides (matrisome-derived peptides are in blue). B. iceLogo plot of cleavage site preference. The arrow indicates the cleaved peptide bond. C. Top ten ADAMTS5 targets. The number of peptides for each target is indicated at the top of each bar. D. Venn diagram showing the overlap between the ADAMTS5, cartilage and synovial fluid degradomes (matrisome-derived peptides only are shown, since ADAMTS5 is a secreted protease). E. Molecules having the most cleavage site overlaps in the cartilage degradome and MMP-13 degradome are shown.
Fig 5: Overlap between forward and reverse degradomics. Proteolytic peptides identified in cartilage degradome (A) and synovial fluid (B) were compared with those generated by MMP13, ADAMTS5 and CMA1. Both unique and overlapping peptides in the various datasets are shown. Peptides found in all three reverse degradomes and also found in the cartilage degradome are shown above the UpSet plot in A. The arrow indicates the cleavage site, and residue numbers flanking the cleavage sites are shown.
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