All animal experimentation was conducted according to National Health and Medical Research Council guidelines (Australia) and with approval from the Howard Florey Institute animal ethics committee.
Human histological studies were conducted in the laboratory of co-author T. Kuhlmann. All human material was sourced from the Neuropathology archives at the University of Muenster. Biopsies were conducted to exclude malignancy, and none of the authors had any part in the clinical management of the patients. Evaluation of neuropathological material was conducted using codified samples and conducted under the governance and with ethics approvals of the University of Muenster.
Animals and reagents
C57B/6 and SJL/J mice were obtained from the Animal Resource Centre (Canning Vale, Western Australia, Australia). The (129sv/C57B/6) Dab2 loxP flanked strain (Dab2fl/fl) was a kind gift from Dr. Johnathan Cooper of the Fred Hutchinson Cancer Research Centre (Seattle, WA) . These mice were backcrossed for 12 generations to generate a Dab2 loxP flanked strain on a C57B/6 background. The Meox2-Cre line was obtained from The Jackson Laboratory (Bar Harbor, Maine) on a mixed background (129sv/C57B/6 backcrossed onto a C57B/6 background for six generations). These mice were further backcrossed on to a C57B/6 background for 8 generations. Dab2fl/fl mice were crossed to Meox2-cre mice to generate animals in which Dab2 was deleted from the embryo, whilst preserving Dab2 expression within extra-embryonic tissue (Dab2 wild-type, Dab2 heterozygote, Dab2 knockout mice).
All chemicals were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise specified. All cell culture plasticware was purchased from Nalgene Nunc International (Rochester, NY). All cell culture media and reagents were purchased from Invitrogen (Carlsbad, CA). All secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA) unless otherwise noted.
Experimental autoimmune encephalomyelitis (EAE) induction
EAE was induced in 8–12 week old male and female mice by subcutaneous injection into the flanks and base of tail of 125μg of MOG35-55 (MEVGWYRSPFSRVVHLYRNGK) or PLP139-151 (HSLGKWLGHPDKF) (Auspep, Australia) emulsified in Complete Freund’s Adjuvant containing 4 mg/ml Mycobacterium tuberculosis H37Ra (Difco, Detroit, MI). Mice also received an intraperitoneal (i.p) injection of 400 ng pertussis toxin (List Biological, Campbell, CA) on days 0 and 3 of induction. Assessments of EAE severity were performed daily according to a 9-point paraplegia scale with 0.5-point increments [43, 44]. Mice that reached grade 3.5 were killed in accordance with ethics committee requirements. EAE cohort 1 comprised Dab2wt/wt (n=32) and Dab2wt/ko (n=37) mice. EAE cohort 2 comprised Dab2wt/wt (n=27) and Dab2ko/ko (n=16) mice. Fewer Dab2ko/ko mouse numbers were available due to the relative difficulty in deriving this genotype.
Unless specified otherwise, all mouse tissue was derived from mice 18 days post-EAE induction.
Non-parametric statistical analyses were used to determine statistical significance of biological phenotypes seen in EAE experiments. The Mann–Whitney Rank Sum Test was used to compare between 2 groups (Dab2wt/wt versus Dab2wt/ko or Dab2wt/wt versus Dab2ko/ko) of EAE challenged animals. Survival was analysed using Kaplan-Meier survival curves followed by Log Rank test statistical analyses.
Microarray analysis of gene expression
Microarray studies were performed on whole spinal tissue from 8 week old SJL/J mice subjected to EAE, and from age and gender matched healthy control mice killed on the same day. All EAE mice were killed at a disease level of severe hindlimb paraparesis or complete hindlimb paraplegia (EAE grade 2.5-3.0). Mice were deeply anesthetized with sodium pentobarbital (100 mg/kg i.p) and intracardially perfused with 20 mls 0.1 M PBS. The spinal cords were removed and immediately snap frozen in liquid nitrogen, and then stored at −80°C until use. RNA was extracted using an RNeasy mini kit (QIAGEN Pty Ltd. Vic, AUS) as per manufacturer’s instructions. Samples were incubated with 600 μl buffer RLT and homogenized using a Dounce homogenizer. The total RNA from spinal cords of two animals were pooled prior to the labeling reaction. A total of three healthy control pair samples (6 mice, n = 3) were compared with a total of five EAE pair samples (10 mice, n = 5). The RNA was then hybridized onto the murine MG U74Av2 microarray, Affymetrix®, Santa Clara CA, following manufactures instructions.
Expression data was analysed using Partek® genomics suite, Missouri, USA. Affymetrix® annotation library MG_U74Av2.na31.annot.csv was used and Data was normalized using RMA (Bolstad et al. 2003) and quantile normalization and adjusted for GC content. Expression values were log transformed using base2 prior to differential expression analysis using a 2 way ANOVA on treatment and batch. Cel files and normalized expression data have been deposited at the Gene Expression Omnibus (GSE44989).
Adoptive transfer EAE
Adoptive transfer EAE was performed as previously described . Briefly, active MOG35-55 EAE was induced in 8 to 12 week old male and female Dab2 knockout (n=5) and Dab2 heterozygous (n=3) donor mice. On day 10 post-immunization, T-lymphocytes were isolated from the draining lymph nodes and spleens of these mice. Cells were cultured in complete DMEM containing 50 ug of MOG35-55 peptide and 20 ng of IL-2, for 48 h at 37C in a 5% CO2 incubator. After this time, the non-adherent T-lymphocytes were collected, washed twice with PBS containing 0.5% FBS, and injected into C57B/6 wild-type recipient mice at a concentration of 2x106 cells in 0.1 ml PBS, i.p, per mouse (transfer ratio was approximately 1 donor to 1 recipient mouse). On the same day, recipient mice were injected with 300 ng pertussis toxin i.p. on the opposite side to the site of T-lymphocyte cell injection. Assessments of disease severity were performed daily for 22 days as described above.
Histology and immunohistochemistry
Mice were anesthetized and intracardially perfused with MT-PBS followed by 4% paraformaldehyde. The lumbar expansions of spinal cords were isolated and equilibrated in 20% sucrose prior to embedding in OCT. Spinal cords were then sectioned at 10 μm intervals and collected onto chrom-alum-coated slides.
To detect murine Dab2, sections were rehydrated in MT-PBS and post-fixed in ice-cold acetone/methanol 50%/50% v/v solution for two minutes. Sections were washed 3x in MT-PBS, and blocked in 10% (v/v) normal goat serum (NGS) in MT-PBS with 0.5% BSA and 0.3% (v/v) TritonX-100 for 1hr at RT. Sections were labelled with rabbit anti-Dab2 (1:100; Santa Cruz Biochemicals, Santa Cruz, CA) alone, or in combination with rabbit anti-NG2 (1:200; Chemicon, Billerica, MA), mouse anti-GFAP (1:500; Chemicon), PE-CD11b antibody (2 μg/ml; CALTAG, Carlsbad, CA), or IgG2b-PE isotype control (2 μg/ml, BD, Franklin Lakes, NJ), rabbit anti-CD3 (1:100, DAKO, Glostrup, Denmark), mouse anti-NeuN (1:500, Chemicon) for 3 hrs at RT. Appropriate fluorescently-labelled secondary antibodies against host species were used at a dilution of 1:500 for 30 minutes at RT. Images were acquired by confocal microscopy (Zeiss LSM5 PASCAL).
Human biopsy and autopsy specimens
This study was approved by the ethics committee of the University of Münster.
Classification of multiple sclerosis lesions
All lesions fulfilled the generally accepted criteria for the diagnosis of multiple sclerosis [46, 47]. Demyelinating activity was classified as described in detail earlier .
Biopsy specimens were fixed in 4% paraformaldehyde and embedded in paraffin. Autopsy material was generally fixed in 10% formalin and tissue blocks containing lesions were embedded in paraffin. Biopsy and autopsy tissues were cut in 4 m thick sections and stained with haematoxylin and eosin, Luxol-fast blue and Bielschowsky’s silver impregnation for lesion classification.
After deparaffinization, antigen retrieval was performed using a slide steamer for 35 minutes with a solution of Tris-EDTA (pH 9.0). After cooling slides on ice and two water rinses, intrinsic peroxidase activity was blocked by incubation with 5% H2O2 in PBS for 20 min. Non-specific antibody binding was inhibited with 10% FCS in PBS for 20 min. For the Dab2 stain, this was followed by incubation with mouse anti-Dab2 IgG1, (BD, 610465) at 1/100, in the same block for 12 hours at 4 degrees. The stain was developed using avidin-biotin immunohistochemistry with a secondary anti-mouse biotinylated Ab and the Vector ABC kit according to the manufacturer’s instructions, followed by the DAB reaction, which was terminated after 8 minutes. Controls omitting the primary Ab showed no staining. The slides were briefly dipped in haematoxylin solution for counter-staining before mounting.
For the APP/CD68 double stain, the sections were first incubated with mouse anti-human APP Ab (Chemicon MAB348) at 1/2000 in block at 4 degrees for 12 hrs, followed by secondary anti-mouse Ab conjugated to alkaline phosphatase, which was visualised using NBT/BCIP. This was followed by incubation with mouse anti-human CD68 (DAKO, M0876) at 1/100 in block for 12 hrs at 4 degrees followed by secondary goat anti-mouse IgG3-HRP (ABD serotec, STAR136P) at 1/200 for one hour, followed by DAB development and brief haematoxylin couterstain followed by mounting.
Morphometry for human sections
Cell numbers and APP positive elements are expressed as an average number per 10 000 mm2 using 40x magnification. Averages were obtained by counting at least 6, and if possible, 10 standardised fields of view per lesion of 10 000 mm2 each defined by an ocular morphometric grid as previously described . Fields for quantitating acute lesions were selected from within the heavily macrophage-infiltrated areas of the biopsies and, for the chronic active lesions, the edge of the lesions containing macrophages/microglia was selected.
cDNA was generated from 1μg of RNA using Taqman Reverse Transcription reagents (Applied Biosystems, Foster City, CA) according to manufacturer’s instructions. Quantitative PCR (qPCR) reactions were performed using Cybergreen PCR mastermix (Applied Biosystems) on a 7500 Fast Real Time PCR System (Applied Biosystems) using the comparative Ct method (Livak & Schmittgen, 2001). qPCR primers were designed using Primer Express 3.0 (Applied Biosystems). Primer sequences were as follows: mouse 18S, forward 5′CGGCTACCACATCCAAGGAA3′, reverse 5′GCTGGAATTACCGCGGCT3′; mouse Dab2 (exon 3 to detect p96 and p67) forward 5′TGTTGGCCAGGTTCAAAGGT3′, reverse 5′GCACATCATCAATACCGATTAGCT3′; mouse IFNγ forward 5′TTGGCTTTGCAGCTCTTCCT3′, reverse 5′TGACTGTGCCGTGGCAGTA3′; mouse iNOS forward 5′GGATCTTCCCAGGCAACCA3′, reverse 5′AATCCACAACTCGCTCCAAGATT3′. qPCR Ct values were normalised to 18S . A dissociation step was performed to ensure that the signal produced was specific to the generation of a PCR product, and not due to primer dimerization.
Statistical significance was tested using the Spearman’s correlation co-efficient to analyse the correlation between EAE grade and fold increase of dab2. One-Way ANOVA with a 95% confidence interval, followed by Tukey’s post hoc comparison was used to test the expression of Dab2 at varying disease grades. Multivariate linear regression analysis was used to analyse the correlation between Dab2 expression and the expression of pro-inflammatory mediators in the spinal cords of Dab2 wild-type and Dab2 heterozygous mice subjected to EAE whilst controlling for EAE score.
Protein lysates (100μg) together with pre-stained protein standards (Bio-Rad, Hercules, CA) were run on 8% Novex Tris-Glycine gels (Invitrogen). Proteins were transferred onto PVDF membranes (Pall Corporation, Port Washington, NY), blocked with 5% skim milk and probed with mouse anti-Dab2 (1:500; BD) at 4°C overnight (O/N). Membranes were washed 4x, followed by incubation with a goat-anti mouse HRP-conjugated secondary antibody (Upstate, Billerica, MA) for 1 hour at RT. Membranes were washed 6x prior to exposure to an enhanced chemiluminescence detection system (Amersham, Fairfield, CT). Signal detection was performed on a FUJIFILM LAS3000 imaging system, using LAS-3000 imaging software.
Histological analysis of EAE disease severity
Inflammation analyses were performed by examining at least four, 10 μm sections per animal taken 50 μm apart to cover a minimum cross-sectional area of 200 μm of the lumber expansion of the spinal cord. Spinal cord area (μm2), the number of discrete immune infiltrates per spinal cord section and the size of each individual infiltrate (μm2) were analysed by an experimenter blinded to the genotype of the mice. Lesions were defined as areas of dense accumulation of Hoechst-positive nuclei both within the white and grey matter. All results are presented as average ± SEM. Statistical significance was tested using One-Way ANOVA with a 95% confidence interval, followed by Tukey’s post hoc test.
Lesion composition analysis was performed on three lesions per transverse section per mouse within the lumbar expansion of the spinal cord. To detect cell-specific markers, 10μm sections were rehydrated in MT-PBS and then blocked in 10% (v/v) NGS in MT-PBS with 0.5% BSA and 0.3% (v/v) TritonX-100 for 1hr at RT. Serial sections were labelled with primary antibodies directed against the microglial marker ionized calcium-binding adaptor molecule-1 (Iba1, 1:1,000; Wako Pure Chemicals, Tokyo, Japan); the astrocytic marker GFAP (1:500; Chemicon); and the oligodendrocyte precursor cell marker NG2 (1:200; Chemicon) in blocking solution at 4°C O/N. Appropriate fluorescently-labelled secondary antibodies against host species were used at a dilution of 1:500 for 1 hour at RT. Sections were also labelled with Hoescht 33342 (1:5,000; Invitrogen) for nucleus detection. Images were obtained using an upright microscope (Zeiss Axioplan 2) at x40 magnification. Images were opened in Adobe Photoshop CS3 and cropped such that the region of interest for each lesion was consistent between cell-specific stains. Regions of interest contained only cells constituting the core of the lesion. Counts of immuno-positive cells with nuclei were determined for each image and expressed as cells/mm2. All counts and analyses were performed blinded to the genotype of the animal. All results are presented as average ± SEM. Statistical significance was tested using One-Way ANOVA with a 95% confidence interval, followed by Tukey’s post hoc test.
Phosphorylated neurofilament heavy chain (pNF-H) enzyme-linked immuno-sorbent assay (ELISA) analyses were performed using a chicken anti-pNF-H antibody (EnCor Biotechnology, Gainesville, FL) as previously described in detail . Statistical significance was tested using a Two-tailed Student’s t-test with a 95% confidence interval.
Isolation and culture of mouse microglia
Primary mixed glial cell cultures were prepared from the brains of P1.5 C57B/6, CD11b-cre Dab2 knockout or Meox2-cre Dab2 knockout mouse pups by exploiting differential adhesion to plastic. In brief, mice were anesthetized with isofluorane, decapitated and brains removed to HBSS (Invitrogen) containing 1mM sodium pyruvate, 10 mM HEPES, 0.14% glucose, 0.3% BSA and 1.16 mM MgSO4. The meninges and choroid plexus were removed; the whole brain was minced and then digested in 0.015% trypsin (w/v) for 15 minutes. The trypsin reaction was stopped with the addition of 0.05% trypsin inhibitor (w/v). Cells were briefly centrifuged and resuspended in DMEM containing 1 mM sodium pyruvate, 0.05% insulin and 10% foetal calf serum (FCS). Cells were triturated to a single cell suspension, plated in PDL-coated 75 cm2 tissue culture flasks and cultured at 37°C/5% CO2. Culture media was changed on days 1 and 7 of culture. Microglia were isolated on day 13 by gentle percussion and collected together with the glial conditioned media (GCM). Cells were briefly centrifuged and plated on 6cm2 tissue culture dishes for 24 h in 50% GCM/50% Macrophage-SFM (Invitrogen), 0.5% FCS to allow cells to quiesce and ramify before experimentation. After 24 h, media was replaced with 100% Macrophage-SFM/0.5% FCS for 16 hours and cultured at 37°C/5% CO2. For assessment of microglial activation cells were either treated with D-PBS (control) or 1 μg/ml LPS for 24 hours prior to RNA isolation.
Isolation and culture of mouse bone marrow macrophages
Mice were given a lethal dose of sodium pentobarbital (100 mg/kg i.p), and immediately following death, the femur and tibia were excised with fine scissors. Using a 23G needle and syringe filled with DMEM (Gibco), the bone marrow was flushed into a tube containing 40 ml of cold DMEM. Cells were pelleted by centrifugation at 350 x g for 5 min, and then resuspended at 1x106 cells/ml in macrophage medium (DMEM, 10% FCS, CSF-1, 100 U/ml penicillin, 100 ug/ml streptomycin, 2 mM GlutaMax-1) and seeded onto a non-coated T75 tissue culture flasks for 3 days in a 5% CO2 incubator at 37°C. After 3 days, non-adherent cells were collected and centrifuged at 350xg for 7 min. The cells were then resuspended in macrophage media and plated at a density of 1.5 million cells/per well onto non-coated 6-well plates (IWAKI). To promote macrophage differentiation, MCSF (2.5 ng/ml) was also added to the media, and the cells were incubated for 4 days. The day before experiments (day 8 following isolation), a full media change was performed (including the addition of MCSF), and cells were left to settle overnight.
Individual statistical tests were performed as described in relevant sections with a minimum alpha level of 0.8. All reported p values are two-tailed and for each analysis p<0.05 was considered significant. Statistical analyses were performed using SigmaStat 2.03 (Systat Software Inc, San Jose, CA). Multivariable linear regression analyses adjusting for EAE disease severity were performed using Stata version 12.0 (StataCorp College Station, Texas).