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: 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 3: Matrilin-2 was cleaved by purified human ADAMTS-4 and ADAMTS-5 specifically. Matrilin-2 peptides were incubated with or without purified recombinant ADAMTS-4 (A), ADAMTS-5 (B) and ADAMTS-1 (C). Western blot analysis of the conditioned medium of Cos-1 cells was performed using an anti-FLAG polyclonal antibody. EDTA (D) or actinonin (E) was added into cell culture. These conditioned media were incubated with or without purified recombinant ADAMTS-4 followed by western blot analysis with an anti-FLAG polyclonal antibody. (F) ADAMTS-4 cleavage of matrilin-2 was dosage dependent. Purified M2L (1 ng) in 10 μL reaction buffer was incubated with purified human recombinant ADAMTS-4 (5 μg/25 μL) at the contents of 0, 1, 2, 4, 8, 12, and 16 μL at 37 °C for 24 h, followed by western blot analysis using anti-flag poly clonal antiboby.
Fig 4: 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 5: Diagram of matrilin-2 and its cDNA constructs. (A) A schematic diagram of matrilin-2 and the amino acid sequence of the unique domain. Alternative splicing region and the antigenic peptide for generation of anti-matrilin-2 antibody were indicated. The numbers correspond to different truncation sites within the domain. (B) Matrilin-2 cDNA constructs with deletions and mutations in the unique domain. M2L(Long) contains the entire unique domain (A838 to E911). M2S(Short) contains the short form of the unique domain without the alternative splicing region (E861 to A879). M2D(Deleted) has the entire unique domain deleted. M2d1, M2d2, M2d3 were matrilin-2 truncated products in which the stop codon was added after E911 (position 1), E888 (position 2), E861 (position 3) in M2L respectively. Position 4 between D and L is the putative unique ADAMTS cleavage site in matrilin-2, while position 1 between E911 and S912 is the second putative ADAMTS cleavage site, similar to the sites in other matrilins.
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