The endogenous proteoglycan-degrading enzyme ADAMTS-4 promotes functional recovery after spinal cord injury

<p>Abstract</p> <p>Background</p> <p>Chondroitin sulfate proteoglycans are major inhibitory molecules for neural plasticity under both physiological and pathological conditions. The chondroitin sulfate degrading enzyme chondroitinase ABC promotes functional recovery after spinal cord injury, and restores experience-dependent plasticity, such as ocular dominance plasticity and fear erasure plasticity, in adult rodents. These data suggest that the sugar chain in a proteoglycan moiety is essential for the inhibitory activity of proteoglycans. However, the significance of the core protein has not been studied extensively. Furthermore, considering that chondroitinase ABC is derived from bacteria, a mammalian endogenous enzyme which can inactivate the proteoglycans' activity is desirable for clinical use.</p> <p>Methods</p> <p>The degradation activity of ADAMTS-4 was estimated for the core proteins of chondroitin sulfate proteoglycans, that is, brevican, neurocan and phosphacan. To evaluate the biological significance of ADMATS-4 activity, an <it>in vitro </it>neurite growth assay and an <it>in vivo </it>neuronal injury model, spinal cord contusion injury, were employed.</p> <p>Results</p> <p>ADAMTS-4 digested proteoglycans, and reversed their inhibition of neurite outgrowth. Local administration of ADAMTS-4 significantly promoted motor function recovery after spinal cord injury. Supporting these findings, the ADAMTS-4-treated spinal cord exhibited enhanced axonal regeneration/sprouting after spinal cord injury.</p> <p>Conclusions</p> <p>Our data suggest that the core protein in a proteoglycan moiety is also important for the inhibition of neural plasticity, and provides a potentially safer tool for the treatment of neuronal injuries.</p

Ablation of KS in microglia accelerated the early phase pathogenesis in a mouse model.

<p>(A) The lifespan of SOD1^G93A was 164.7±18.2 days (n = 43), while that of SOD1^G93AGlcNAc6ST-1^−/− was 158.7±13.2 days (n = 76) (p = 0.007). (B) Body weight loss of SOD1^G93A began at 124.7±12.2 days, while it began at 118.4±11.9 days in SOD1^G93AGlcNAc6ST-1^−/− (p = 0.005). A decrease in rotarod performance at (C) 15 rpm and (D) 20 rpm of SOD1^G93A began at 141.4±18.2 days and 121.6±24.5 days, respectively. In SOD1^G93AGlcNAc6ST-1^−/− mice, these onset times were accelerated to 125.2±14.2 (p<0.001) days and 112.5±18.1 days (p = 0.003), respectively.</p

Expansion of M2 microglia during the early disease phase was suppressed in SOD1^G93AGlcNAc6ST-1^−/− mice.

<p>The spinal cord sections of SOD1^G93A mice and SOD1^G93AGlcNAc6ST-1^−/− mice at 15 and 24 weeks were stained with KS (A, E, I, M), M2 marker CD206 (B, F, J, N), and microglia marker Iba1 (C, G, K, O). Arrows, KS^–CD206⁺ microglia; arrowheads, KS⁺CD206⁻ microglia. Bars, 50 µm.</p

M1 microglia expanded as the disease progressed.

<p>The spinal cord sections of SOD1^G93A mice and SOD1^G93AGlcNAc6ST-1^−/− mice at 15 and 24 weeks were stained with KS (A, E, I, M), M1 marker CD86 (B, F, J, N), and microglia marker Iba1 (C, G, K, O). Arrows in A-H, KS⁺CD86⁺ microglia; arrows in J-P, KS^–CD86⁺ microglia; arrowheads, KS⁺CD86⁻ microglia. Bars, 50 µm.</p

CD206 was expressed in a subpopulation of microglia distinct from KS+CD86- cells.

<p>Sequential thin sections were subjected to immunohistochemical analyses (A). The spinal cord sections of SOD1^G93A mice at 15 weeks were stained with KS (B), CD86 (C) and CD206 (D). Arrows, KS⁺CD86⁻ microglia; arrowheads, KS^–CD206⁺ microglia. Bars, 50 µm.</p

Transient enhancement of the expression of M2 markers was diminished in SOD1^G93AGlcNAc6ST-1^−/− mice.

<p>The temporal mRNA expression profiles of M1 [CD86 (A), IL-1β (B), TNF-α (C) and NOX2 (D)] and M2 [Arginase1 (E), CD206 (F), Ym1 (G) and IL-4 (H)] markers were examined by quantitative RT-PCR. Gray columns, SOD1^G93A mice; black columns, SOD1^G93AGlcNAc6ST-1^−/− mice. Error bars, SE. **p<0.01, *p<0.05 (n = 3).</p

KS was expressed in a subpopulation of microglia.

<p>(A-I) The SOD1^G93A mice lumbar spinal cords at 18 weeks were stained by anti-KS (B, E, H), anti-Iba1 (microglia marker; A–C), anti-GFAP (astrocyte marker; D–F), and anti-NG2 (oligodendrocyte precursor marker; G–I) antibodies. Merged images are shown in C, F and I. Bars, 50 µm. Arrows, KS⁺Iba1⁺ cells; arrowheads, KS^-Iba1⁺ cells. (J) A representative profile of KS expression in CD11b⁺ cells. The red line indicates a negative control that does not contain the primary antibody, and the blue line indicates a stained sample. (K) The quantitative data of mean fluorescence intensity (MFI) of KS (n = 3). Error bars, SE. **p<0.01. N.S., not significant. (L) The biosynthesis of KS. (M) mRNA expression of the enzymes involved in KS biosynthesis was examined by quantitative RT-PCR using SOD1^G93A (gray columns) and their age-matched non-Tg (white columns) mice at 24 weeks. GAPDH was used as the internal control. Error bars, SE. ** p<0.01, * p<0.05 (n = 4). N.S., not significant.</p

The primer sequences using quantitative RT-PCR of M1 and M2 markers.

<p>The primer sequences using quantitative RT-PCR of M1 and M2 markers.</p