The following were obtained commercially: acrylamide (Sigma-Aldrich, St. Louis, MO); Neurobasal medium (Invitrogen, Carlsbad, CA); fetal bovine serum (FBS) (Hyclone, Logan, UT); L-glutamine (Invitrogen, Carlsbad, CA); B27-serum free supplement (Invitrogen, Carlsbad, CA); nerve growth factor (NGF) (Sigma-Aldrich, St. Louis, MO); trypsin (Invitrogen, Carlsbad, Ca); collagenase I (Worthington, Lakewood, NJ); β- III tubulin (Promega, G7121); anti-MAG (R & D Systems, AF538); anti—Fc (R & D Systems, AF2049); anti-tyrosinated tubulin (Sigma-Aldrich, T9028); anti-detyrosinated tubulin (Chemicon, AB3201); MAG-Fc (R & D Systems, 538-MG); Fc (R & D Systems, AF2049); recombinant active MMP-7 (Calbiochem/EMD Biosciences , Cat. No. 444270). Mutant proteins MAG-Fc and OMgp were provided by Dr. Guo-Li Ming.
MAG knockout founder mice, kindly provided by Dr. John Roder, University of Toronto, Ontario, Canada, were constructed by disruption of exon 5 of the MAG gene as previously reported (Ng et al., 1996
). The strain provided (identical to that available from the Jackson Laboratory, Bar Harbor, ME) was on a C57BL/6, 129 inbred strains and CD1 Random bred strain. To enhance comparisons between mutant strains, mutant mice were repeatedly back-crossed onto a C57BL/6 background to >99% strain purity (Pan et al., 2005
NgR1 knockout and wild-type liter-mate mice, kindly provided by Dr. Stephen Strittmatter, Yale University School of Medicine, New Haven, CT, were constructed by disruption of exon 2 of the NgR gene as previously reported (Kim et al., 2004
). The strain provided was on a C57BL/6 background.
Mice were anesthetized with chloral hydrate and perfused through the ascending aorta with freshly prepared 4% paraformaldehyde in 0.1 M sodium phosphate (pH-7.4). The C-5 spinal cord, sciatic nerves, and distal tibial nerves were harvested from groups of mice at 6, 12, and 15 months of age. Five mice were used for each group. The tissues were further fixed in 4% paraformaldehyde / 3% glutaraldehyde in Sorenson's buffer overnight at 4°C, post-fixed in OsO4, and embedded in Epon-Araldite resin.
Cross sections (1-μm thick) were stained with toluidine blue for analysis under light microscopy with a 60X or 100X oil immersion objective lenses using a stereotactic imaging software (Stereo Investigator version 5). Every myelinated fiber in easily defined regions of the CNS and PNS was counted (Supplemental Fig 1
). The resulting data were free of counting biases that could affect both random and systematic (stereologic) schemes of number of axon counting. Actively degenerating fibers were defined as fibers at different stages of Wallerian-like degeneration, such as myelin figures and ovoids. Results from each studied group were compared by the two-tailed Students t-test. p<0.05 was considered significant.
Groups of five 6-week-old male mice were treated with acrylamide by adding acrylamide to the drinking water at 400 ppm. Control mice drank regular water. Five mice were housed in each plastic cage throughout the experimental period. Experimental procedures followed the principles in the “Use of Animals in Toxicology” and NIH guidelines (“Guide for the Care and Use of Laboratory Animals,” NIH Publication No. 86-23, 1985).
Groups of five mice were trained one week prior to acrylamide treatment on 3 consecutive days by performing the Rotarod at a constant speed of 4 rotation per minute (rpm) on day 1 and 8 rpm on day 2 and 3 for 3 minutes (pre-training), followed by an accelerating protocol in which the speed of rotation was initially set at 4 rpm and accelerated an additional 4 rpm every 30seconds to a maximum of 40 rpm. Three trials were performed on each mouse on each of 3 consecutive days with approximately 30 minutes rest between trials. Following initiation of acrylamide treatment, the mice were further trained for another 3 consecutive days. Two weeks after acrylamide intoxication, mice were again pre-trained and tested for 3 days, 3 trials per day. Unlike untreated mice, most MAGKO mice fell off the rod even during pre-training (mice were not returned to the rod more than twice during pre-training).
To analyze data, we recorded the speed and duration during which the mouse stayed on the rod. Results of all 3 trials on day 3 of testing were pooled to generate a mean and SEM. (n= 15). Results were compared by the two-tailed Students t-test. Values of p<0.05 were considered significant. The tester was blinded to the treatment. No mice or values were excluded.
Nerve conduction studies were performed on five wild-type and five MAG knockout mice at 6-8 weeks of age. The mice were anesthetized with isofluorane using a nose cone. Body temperature was maintained on a warm blanket to keep the surface temperature between 32° and 38°C prior to the taking of measurements. All compound motor action potentials (CMAP) measurements were performed with a PowerLab signal acquisition setup (ADInstruments). The CMAPs were measured by stimulation with subdermal needle electrodes placed near the sciatic nerve at the sciatic notch. Recording electrodes were placed in the tibial nerve-innervated intrinsic foot muscles in the plantar surface. Recordings were made with supramaximal stimulation. We determined the latencies, negative peak amplitudes, and durations of the sciatic compound muscle action potentials as well as the F-wave latencies and durations. The distance between stimulation sites, determined by calipers, was also used to calculate conduction velocities. CMAP measurements were obtained at the initiation of acrylamide intoxication and 2-1/2 weeks after intoxication.
DRG explant culture
P4-5 dorsal root ganglia (DRG) were removed from Sprague-Dawley rat pups and maintained in neurobasal medium containing 2M L-glutamine, 2% B27-serum free supplement and 50-100ng/ml nerve growth factor (NGF). Explant cultures were allowed to grow to a mature and maintenance state for 5 days, creating a lush outgrowth of neurites. This method of allowing neuritic outgrowth extension to proceed before addition of MAG and/or a neurotoxin test their effect on established neurites as opposed to their effect on primary neuritic outgrowth. The neurons were examined under phase contrast microscopy, and the average neurite length were quantified by using IMAGE J, a public domain image processing program (http://rsb.info.nih.gov/ij/
). The axonal lengths of all axons were measured from the explant's border to the tips of intact axons. An average of all axonal lengths for each explant was then calculated. At least 8-10 explants were used for each test conditions.
In experiments involving MAG-CHO cells, DRG explants were allowed to grow to a mature and maintenance state for 5 days. MAG-CHO cells were then added to the culture and allowed to grow for additional 48 hours before the effect of MAG-CHO cells on axonal protection was examined.
A tetracycline repressible MAG system was generated in Chinese hamster ovary (CHO) cells as described previously (Milward et al., 2008
). Several clones were derived and simultaneously assessed for expression of MAG and DsRed2, tetracycline repressibility of MAG and membrane-bound expression of MAG. Addition of the tetracycline analog doxycycline at 1 μg/ml caused complete inhibition of MAG expression. In the present studies, MAG was allowed expressed throughout the experiments.
Results from each studied group were compared by the two-tailed Students t-test. p<0.05 was considered significant.
Dissociated P4-5 DRG and Cerebrocortical Neurons
P4-5 dorsal root ganglia (DRG) and cortical neurons were removed from 4 animals and incubated with 0.25% trypsin. The digestion solution for post-natal DRG neurons also included 0.3% collagenase I. After 30 minutes, digestion was stopped with 10% FBS containing L-15 medium, and cells were plated at 10,000/cells per well. Cultures were maintained in neurobasal medium containing 1% FBS, 2M L-glutamine, 2% B27-serum free supplement and 50-100ng/ml nerve growth factor (NGF). Neuronal cultures were allowed to grow to a mature and maintenance state for 5-7 days, creating a lush outgrowth of neurites. The average neurite lengths of 50 neurons immunostained for class III β-tubulin were quantified under fluorescent microscopy using Image Lab. Each experiment was performed in triplicate. Results from each studied group were compared by the two-tailed Students t-test. p<0.05 was considered significant.
Dissociated DRG and cortical neurons were plated onto collagen-coated tissue culture dishes in the middle of a three-compartment chamber. Compartmentalized cultures were prepared as previously described (Campenot, 1982
). Cells were maintained for the initial 2-7 days in growth medium containing cytosine arabinoside (10 uM) to eliminate Schwann cells and non neuronal, dividing cells. After growing for 7 days, axons from the central chamber extended into the side chambers. The central compartment contained the cell bodies and proximal axons (M), while the side compartments contained the distal axonal processes and axon terminals (D). Treatment mediums were added to the side chambers, allowing selective delivery of the stimuli to axons. The average axonal lengths of 50 neurons immunostained for class III β-tubulin were quantified under fluorescent microscopy using Image Lab. Axonal length was measured from the edge of the chamber to the intact axonal tip. Each experiment was performed in 5-8 culture plates. Results from each studied group were compared by Students t-test. Values of p<0.05 were considered significant.
Mutant MAG peptide with KGE domain
Mutation on MAG (from RGD to KGE) was generated by site directed mutagenesis (Stratagene) as described by the manufacturer. Mutant MAG-Fc with KGE domain was then transfected into 293 Ebna cells. The proteins were collected from the media and affinity purified using protein A sepharose. Wild-type MAG-Fc with RGD domain also were expressed and purified in a similar manner.
MAG was extracted from purified myelin membranes using mild detergent and was adsorbed to culture surfaces as described (Mehta et al., 2007
). Briefly, myelin was purified from brains freshly dissected from adult Sprague-Dawley rats or adult wild-type or MAG-null mice and stored at -70°C prior to use. Myelin membranes were suspended at 1 mg protein/ml in extraction buffer (0.2 M sodium phosphate buffer (pH 6.8), 0.1 M Na2
, 1 mM EDTA, 1 mM DTT, protease inhibitor mixture (Sigma), and 1% octylglucoside), incubated at 4 °C for 16 hours with gentle agitation, then centrifuged at 100,000 × g
for 1 hours at 4°C. The supernatant was collected and diluted with an equal volume of detergent-free buffer and an aliquot (50 μl) was added to each well of a PDL-coated 96-well plate. After 4 hours at ambient temperature, the plate was washed with Dulbecco's phosphate-buffered saline (PBS) and then with the culture medium appropriate to the cell type prior to plating freshly prepared cells. DRG and cortical neurons were grown on the substrate as described previously (Mehta et al., 2007
After 48 hours cultures were washed with PBS, fixed overnight with 2% paraformaldehyde in PBS, then permeabilized using 0.1% Triton X-100 in PBS. DRGN were immunostained with anti-neuronal class III β-tubulin monoclonal antibody (TUJ1, 1:2000, Covance, Berkeley, CA) followed by Cy3-conjugated anti-mouse IgG (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA). After washing, multiple random fields were captured for image analysis using a Nikon TE300 epifluorescent microscope fitted with a Photometrics CoolSNAP HQ2 camera (Roper Scientific, Duluth, GA).
DRG neurons on control substrata extended long, thick neurites when grown in control conditions. In contrast, DRG neuronal cultures grown in the presence of vincristine (without myelin substrata) have neurites that progressively degenerate distally where they become fragmented, comparatively thinner, and morphologically distinguishable from neurites in cultures without vincristine. To differentiate the integrity of neurites, we developed an image analysis protocol using NIS-Elements software (Nikon, Melville, NY) to detect the intact, non-fragmented axons. The protocol selected neurites that were >150 μm long, and subtracted out the portion of neurites that are fasciculated. Neuritic lengths were summed and divided by the total number of DRG neuronal cell bodies to provide a single value for the presence of healthy unfasciculated neurites.
For each experimental condition 4-5 random images from each of 5-10 independent wells from an average of 3 independent experiments were analyzed. Data are presented as the mean ± standard error of the mean. Results were compared using the two-tailed Students t-test. Values of p<0.05 were considered significant.
Cell monolayers were scraped and lysed in a detergent-containing buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 2 mM sodium orthovanadate, 10 mM NaF, 10 mM sodium pyrophosphate, 1% IGEPAL, 1 mM phenylmethylsulfonyl fluoride) with protease inhibitor cocktail (Roche, Mannheim, Germany). Total protein (10 μg) was loaded on 4-12% Tris/Glycine gradient gels and transfered to nitrocellulose membranes (Novex, San Diego, CA, USA). Membranes were blocked in 10% bovine serum albumin (BSA) in Tris-buffered saline (TBS)/0.05% Tween-20 (TBS-T) for 1 hour at room temperature, and then incubated with the primary antibodies and their appropriate peroxidase-conjugated secondary antibodies for varying times. Blots were developed using enhanced chemiluminescence (ECL) (NEN Life Science Products, Boston, MA, USA). Antibodies for specific proteins are listed in the Reagents section.
Skin tissue was fixed for 12-18 h in 2% paraformaldehyde/lysine/periodate fixative followed by cryoprotection. The tissue were sectioned with a sliding microtome into 50-mM thick free-floating sections. Sections were stained with monoclonal antibodies to neurofilament (NF160 1:200, Chemicon) and developed with chromogens as previously described (Ebenezer et al., 2007