Background
Following neuronal injury, damaged axons may regenerate or spared axons may sprout, resulting in synaptic reconnection and reconstruction. However, such plasticity after neuronal injury does not occur extensively in the adult mammalian central nervous system (CNS). This is due to a lack of growth-promoting factors, the poor intrinsic regenerative capacity of CNS neurons and the emergence of inhibitory factors after injury [
1‐
7].
Chondroitin sulfate (CS) proteoglycans (CSPGs) are glial scar-associated inhibitors. The inhibitory function of CSPGs on axonal outgrowth is primarily due to their covalently attached CS-glycosaminoglycans. For example, the CS-degrading enzyme chondroitinase ABC (C-ABC) enhances axonal regeneration/sprouting after CNS injury and consequently promotes functional recovery [
8,
9]. Supporting these data, CSPGs strongly inhibit neurite outgrowth
in vitro, and C-ABC reverses it [
7]. In addition to CSPGs, we found that keratan sulfate proteoglycans (KSPGs) inhibit axonal regeneration/sprouting after injury [
10,
11]. Thus,
in vivo neurite outgrowth after a cortical stab wound is enhanced in mice deficient in 5D4-reactive KS in the CNS [
10]. These mice also exhibit better motor function recovery after spinal cord injury (SCI) [
11]. Furthermore, we found that proteoglycans including both CSPGs and KSPGs inhibit neurite outgrowth
in vitro, and the KS-degrading enzyme keratanase II reverses this inhibition [
11]. These data collectively suggest that glycosaminoglycans (GAGs), such as CS and KS, in a proteoglycan moiety play a central role in proteoglycan-mediated inhibition of neural plasticity after injury.
As both C-ABC and keratanase II are of bacterial origin, repeated use of these enzymes may give rise to adverse effects, such as undesirable generation of antibodies, making their application to therapy limited. Therefore, it would be ideal if we could use an endogenous mammalian enzyme that inactivates the proteoglycans' inhibitory function. However, a fundamental question must be answered before using a mammalian enzyme for this purpose. Namely, since proteoglycan is composed of a core protein and GAGs, it should be determined whether the core protein, in addition to the GAGs, in the proteoglycan moiety is essential for the proteoglycan-mediated inhibition.
Based on the above background, we investigated the effects of a proteoglycan-degrading enzyme, ADAMTS-4 (a disintegrin and metalloproteinase with thrombospondin motifs-4; also designated aggrecanase 1, which stands for an enzyme degrading aggrecan, a common CS/KSPG in the brain and cartilage), on neural plasticity after SCI in this study. As ADAMTS-4 is known to degrade the core protein of some CSPGs, we may determine the importance of the core protein on axonal regeneration/sprouting. Here, we demonstrate that ADAMTS-4 reverses the proteoglycan-mediated inhibition of neurite outgrowth in vitro, and promotes functional recovery after SCI.
Methods
Spinal cord surgery
Adult female Sprague-Dawley rats weighing 200 to 230 g were used in this study. The animals were anesthetized by intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). After Th10 laminectomy, we exposed the dura mater and induced injury using a force of 200 kydn with a commercially available spinal cord injury device (Infinite Horizon Impactor; Precision Systems & Instrumentation, Lexington, KY, US). Immediately after the spinal cord contusion, we performed a Th11 partial laminectomy and inserted a thin silicone tube with an osmotic mini-pump into the subarachnoid cavity under a surgical microscope. The osmotic mini-pump (Alzet pump model 2002 (Durect Corporation., Cupertino, CA, US): 200 μl solution, 0.5 μl/hour, 14-d delivery) was filled with recombinant ADAMTS-4 (100 nM) (R&D Systems, Minneapolis, MN, US), C-ABC (0.05 units/200 μl) (Seikagaku Corporation, Tokyo, Japan) or PBS (as a vehicle control). The tube was sutured to the spinous process to anchor it in place and the minipump was placed under the skin on the animal's back. Afterward, the muscles and skin were closed in layers. The bladder was compressed by manual abdominal pressure twice a day until bladder function was restored. Food was provided on the cage floor, and the rats had no difficulty reaching their water bottles. All animals were given antibiotics in their drinking water (1.0 ml Bactramin (Roche, Basel, Switzerland) in 500 ml acidified water) for two weeks after SCI. All animals were treated and cared for in accordance with the Nagoya University Graduate School of Medicine guidelines pertaining to the treatment of experimental animals.
Western blot analysis
To determine the ADAMTS-4 expression in the injured spinal cord of the rats, the diffusion of His-tagged ADAMTS-4 infused into the injured spinal cord tissue and the degradation of brevican and neurocan by recombinant ADAMTS-4 enzyme, tissue extracts were analyzed by SDS-PAGE/Western blot with anti-ADAMTS-4, anti-His-Tag, anti-brevican, and anti-neurocan antibodies. Samples of the supernatant fraction were collected after centrifuging at 10000 g for 15 minutes and were separated by electrophoresis on 5% SDS-PAGE. Proteins were then blotted onto nitrocellulose membranes. Blots were blocked with 5% fat-free dry milk in PBS for 60 minutes and incubated overnight at 4°C with the primary antibody (anti-ADAMTS-4(1000 × dilution; Santa Cruz Biotechnology, Santa Cruz, CA, US), anti-His-Tag (1000 × dilution; MBL, Nagoya, Japan), anti-brevican (1000 × dilution; BD Biosciences, San Jose, CA, US), and anti-neurocan (1000 × dilution; clone 1G2, Seikagaku Corporation) ) in PBS containing 0.3% Triton X-100. They were washed and then were incubated with a second antibody (horseradish peroxidase-conjugated anti-goat and anti-mouse immunoglobulin G (IgG; 5000 × dilution; Jackson ImmunoResearch, West Grove, PA, US)) in PBS containing 0.3% Triton X-100 at room temperature for 60 minutes. Anti-β-actin antibody (1000 × dilution; Sigma, St. Louis, MO, US) was also used as indicated. Bound antibodies were visualized with an ECL and ECL-plus Western blotting detection kit (GE Healthcare, Buckinghamshire, UK).
Fluorescent assay of ADAMTS-4 activity
To measure ADAMTS-4 activity in the rat spinal cord, we used the SensoLyte® 520 Aggrecanase-1 Fluorometric Assay Kit (AnaSpec, Fremont, CA, US). A tissue sample from the rat spinal cord was diluted in 50 μl of assay buffer in a 96-well white plate (Sumitomo Bakelite, Tokyo, Japan). Then, 50 μl of aggrecanase substrate solution was added into each well and incubated for 60 minutes at room temperature. Fluorescence was measured at 30°C using a POWERSCAN 4 (DS Pharma Biomedical, Osaka, Japan) equipped with a 490-nm excitation filter and a 520-nm emission filter.
To evaluate the thermo-stability of ADAMTS-4, the enzyme (10 nM) was incubated in vitro at 37°C. After two weeks incubation, the enzyme activity was measured.
Cell culture
Sprague Dawley rats were killed on postnatal days 7 to 9, and the cerebella were collected. The meninges were carefully removed with fine forceps and the remaining tissues were minced and digested using a Papain Dissociation System (Worthington Biochemical, Lakewood, NJ, US). Dissociated cells were applied to a 35/60% two-step Percoll gradient and centrifuged at 3000 g for 15 minutes. Cerebellar granule neurons at the interface were collected. Cells were suspended in neurobasal medium (Invitrogen, Carlsbad, CA, US) supplemented with 2% B27 (Invitrogen), 2 mM glutamine, an additional 20 mM KCl, 50 U/ml penicillin, and 50 μg/ml streptomycin.
Primary cultures of cerebral cortical astrocytes were prepared from newborn Sprague-Dawley rats as previously described [
12,
13]. Briefly, forebrains were removed aseptically from the skulls, the meninges were excised carefully under a dissecting microscope and the cortices were isolated. The small tissues obtained by mincing the cortices were cultured in flasks in DMEM containing 10% fetal bovine serum (FBS), then incubated at 37°C in a humidified atmosphere containing 5% CO
2. The culture medium was renewed every three to five days. Experiments were performed on confluent 30-day-old cultures. More than 95% of the obtained cells were glial fibrillary acidic protein (GFAP)-positive. Astrocytes were plated at a density of 5 × 10
5 cells per 3.5-cm dish.
Microglia-enriched cultures were obtained using the method of Giulian et al. [
14]. Briefly, small pieces of tissues were obtained by mincing the cortices as described above for the astrocyte primary culture and then were cultured in flasks in Mi-medium (DMEM, 10% FBS, 0.2% glucose and insulin 5 μg/ml). The mixed glial culture grown for 21 days was subjected to shaking at 200 rpm on a gyratory shaker for 30 minutes. The detached cells (mainly microglia) were reseeded in fresh culture flasks, and after two hours any contaminating oligodendrocyte progenitors were detached with Tris-buffered saline containing 1 mM ethylenediaminetetraacetic acid (EDTA). This procedure routinely provides a firmly attached homogeneous population of microglia. Microglia were cultured in DMEM containing 10% FBS. More than 95% of the obtained cells were found to be Iba1-positive. Microglial cells were plated at a density of 1 × 10
5 cells per 3.5-cm dish.
RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR)
The total RNA of the cells was isolated using an RNeasy Tissue Mini Kit (Qiagen, Valencia, CA, US) according to the manufacturer's instructions. One microgram of purified total RNA was transcribed using SuperScript III First-Strand Synthesis Super Mix (Invitrogen). The cDNA products were used for the polymerase chain reactions (PCRs), which were performed using a Veriti 96-well Thermal Cycler (Applied Biosystems, Foster City, CA, US) according to the following protocol: 35 cycles of denaturing at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and elongation at 72°C for 60 seconds. The rat ADAMTS-4 primers were 5'-ctacaaccaccgaaccgac-3' (forward) and 5'-tgccagccaccagaactt-3' (reverse). The rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers were 5'-tatgactctacccacggcaag-3' (forward) and 5'-tgcattgctgacaatcttgag-3' (reverse).
PG degradation assay by ADAMTS-4
Whole brains were isolated from adult female Sprague-Dawley rats. Tissues were homogenized in PBS containing 10 mM N-ethylmaleimide and protease inhibitor mixtures (Nacalai Tesque, Kyoto, Japan) using a Dounce-type homogenizer. Homogenates were centrifuged at 24000 g for 30 minutes and supernatants were applied to diethylaminoethyl (DEAE) Sepharose (GE Healthcare). Samples were washed three times with wash buffer (50 mM Tris-HCl, pH 7.5, 2 M urea, 0.25 M NaCl, 20 mM EDTA, 0.2 mM PMSF, 1 mM N-ethylmaleimide), and the proteoglycans were eluted with 2 M NaCl. The eluent was concentrated using a size-exclusion spin column (molecular weight cutoff, 100 kDa), and the protein concentration was determined using a Micro BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, US). Purified PG samples were incubated with 1 μM recombinant human ADAMTS-4 and ADAMTS-13 (R&D Systems) (diluted in 10 mM Tris-HCl, 0.15 M NaCl, and 10 mM CaCl2) for 3 hours at 37°C. Controls included samples with ADAMTS-4 that were inactivated by heating at 95°C for 30 minutes. After a 3 hour incubation period, 1 M dithiothreitol (DTT) SDS-PAGE sample buffer was added to the samples, and then the samples were heated at 95°C for 5 minutes and subjected to SDS-PAGE and Western blotting. Membranes were probed with mouse anti-brevican, anti-neurocan and anti-phosphacan (1000 × dilution), and the primary antibody was detected with anti-mouse, anti-rabbit IgG conjugated to horseradish peroxidase. Bound antibodies were visualized with an ECL Western blotting detection kit.
Neurite outgrowth assays
Sprague Dawley rats were killed on postnatal days 7 to 9, and the cerebella were collected. The meninges were carefully removed with fine forceps and the remaining tissues were minced and digested using a Papain Dissociation System. Dissociated cells were applied to a 35/60% two-step Percoll gradient and centrifuged at 3000
g for 15 minutes. Cerebellar granule neurons at the interface were collected. Cells were suspended in neurobasal medium supplemented with 2% B27, 2 mM glutamine, an additional 20 mM KCl, 50 U/ml penicillin, and 50 μg/ml streptomycin. Four-well chamber slides (NUNC, Roskilde, Denmark) were coated with 20 μg/ml poly-L-lysine (PLL; Sigma) and left overnight at 4°C and then were coated with chick brain proteoglycans (Millipore Bioscience Research Reagents, Temecula, CA, US) and left for 4 hours at 37°C. If indicated, proteoglycans were treated with 10 nM ADAMTS-4 or 200 mU/ml C-ABC in PBS at 37°C. Cerebellar granule neurons were seeded onto four-well chamber slides at 2.0 × 10
5 per well. Twenty-four hours after seeding, the neurons were fixed with 4% paraformaldehyde/PBS and stained with anti-neuron-specific β-tubulin (Covance, Princeton, NJ, US) to visualize neurites. Neurite lengths were measured from at least 100 neurons that had neurites longer than twice the cell body diameter, per condition from duplicate wells, and quantified as described previously [
15]. The number of adherent cells was counted under 200 × magnification (six fields).
Behavioral test
The locomotor performance of animals was analyzed using the Basso, Beattie and Bresnahan scale (BBB) open-field score for eight weeks, since the BBB has been shown to be a valid locomotor rating scale for rats. The evaluations were made by two blind observers for all analyzed groups. Briefly, the BBB is a twenty-one-point scale that provides a gross indication of locomotor ability and determines the phases of locomotor recovery and features of locomotion. The BBB score was determined for nine rats in each group.
Immunohistochemistry
Rats were perfused transcardially under deep ether anesthesia with buffered 4% paraformaldehyde. The spinal cords were removed, postfixed in 4% paraformaldehyde overnight and cryoprotected in buffered 30% sucrose during the subsequent night. Tissues were cut into 20 μm sections with a cryostat and mounted on glass slides. Sections were blocked in PBS containing 3% BSA. Sections were then incubated with 5-hydroxytryptamine (5-HT) antibody (100 × dilution; Immunostar, Hudson, WI, US) in a blocking solution overnight at 4°C in PBS containing 3% BSA. After rinsing in PBS, the sections were incubated with the secondary antibody (100 × dilution; Alexa 488 conjugated anti-rabbit antibody; Invitrogen) for 60 minutes at room temperature. Subsequently, the sections were rinsed in PBS, mounted with FluorSave (Calbiochem, San Diego, CA), and examined by an Olympus model BX41 microscope fitted with the appropriate filters.
Morphometry
The epicenter of a lesion was determined by hematoxylin and eosin staining of several of the serial 20 μm sections. All the cross-sectional image analyses were performed using spinal cord samples from positions 5 mm caudal to the lesion site. Mean values for each animal were then compared. Light intensity and thresholding values were maintained at constant levels for all analyses by a computer-driven microscope stage (MetaMorph Offline version 6.3 r
2; Molecular Devices, Sunnyvale, CA, US). The amounts of axonal outgrowth of the wound area were assessed by counting signals visualized by staining with anti-5-HT antibody, for 650 × 860 μm2 counting frames around a lesion. Statistical analyses were performed for five rats for each experimental group.
Statistical analysis
Using SPSS (SPSS Inc., Chicago, IL, US), data for the BBB score were analyzed by repeated measures analysis of variance (ANOVA) with a post hoc Bonferroni test. Data were statistically analyzed using an unpaired two-tailed Student's t-test for histological assessment for the area of 5-HT-positive fiber. In all statistical analyses, values of P < 0.05 were considered to indicate significance. When gathering data for the statistical analyses, the investigators were blinded to each group in all procedures.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
RT, SI, and RS performed in vivo experiments. RT, TN, TO and AM carried out in vitro experiments. RT, SI, YM, NI and KK designed the study. RT and KK wrote the manuscript. All authors read and proved the final manuscript.