DNA amplification for cloning

For cloning the different constructs described in listed in the previous section (Table 2.8), we amplified DNA fragments by PCR using with Phusion polymerase (2X Phusion High-Fidelity PCR Master Mix with HF Buffer, Finnzymes).

Table 2.8:PCR with Phusion Polymerase mix

Component Amount

2X Phusion mix containing: 25µL

-0.2 mM dNTPs

-1 X Phusion HF Buffer

-1 X Phusion High-Fidelity DNA Polymerase

Primer 1 (10 pmol/µL stock) 2.5µL Primer 2 (10 pmol/µL stock) 2.5µL

DNA (10 ng/µL) 1µL

DMSO 1,5 ul (3%)

ddH2O (add to 50µL) 17.5µL

3% DMSO is suggested as a PCR additive to facilitate the denaturation of templates rich in GC. The general PCR protocol used was:

Table 2.9:PCR protocol for cloning using Phusion polymerase

Component Temperature Time

Plasmids and PCR products were run in 1-2% Agarose gel in 1×TAE buffer with 0.5 mg/mL ethidium bromide (Sharp et al., 1973) for the separation and visualization of DNA frag-ments. Chambers assembled by the Feinmechanik Service Department at the Max Planck Institute for Experimental Medicine were used for electrophoresis in 1×TAE buffer. The DNA was visualized by transillumination using Intas Gel Documentation System (Intas Science Imaging Instruments GmbH).

After agarose gel electrophoresis, DNA fragments were extracted with NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel) following manufacturer’s instructions, and eluted in 15 µL.

DNA concentration determination

The concentration of DNA fragments and plasmids was determined measuring the ab-sorbance at 260 nm of 1µL of DNA diluted in 49µL of H₂O, using a BioPhotometer plus (Eppendorf). The formula used was:

Concentration of DNA (µg/mL) =A260×dilution factor×50µg/mL (2.1) 2.2.1.3 DNA digestion with restriction enzymes

Digestion of DNA fragments were done using restriction enzymes, due to their ability to cleave DNA fragments according to specific sequences (Arber and Linn, 1969). Restriction enzymes were purchased from New England Biolabs and Fermentas. A typical reaction was prepared as follows:

Table 2.10:Digestion with restriction enzymes

Component Reaction Vector Control

10X buffer 3µL 3µL 3µL

DNA 4µg (1µg/µL Midiprep) or 15µL PCR elution 4µg (Midiprep) 4µg (Midiprep)

BSA 10X 3µL 3µL 3µL

Water 18-19µL/8µL 18-19µL 19-20µL

Enzyme(s) 1µL each 1µL each

The mixture was left for 1 hour generally at 37℃unless specified for a particular enzyme.

When required, vector dephosphorylation was performed by adding, after digestion, 3 µL 10X Antarctic phosphatase buffer and 1µL Antarctic phosphatase (New England Biolabs) to the digestion mix, leaving at 37℃ for 1 hour and heat inactivating for 5 minutes at 65℃ .

2.2.1.4 DNA Ligation

Digested fragments were ligated using T4 DNA ligase (Fermentas) as follows:

Ligation reaction

The mix was incubated for at least 1h at RT, or overnight at 4°C and used for bacterial transformation.

2.2.1.5 Transformation of E. coli

For transformation, 50-100µL of DH5_alphaE. coli (Subcloning Efficiency DH5_alpha, Invit-rogen) were thawn on ice. 5-10 µL of ligation (or 0.5 µg of plasmid DNA for retransfor-mation) were added, mixed gently and incubated 30 min on ice. The mix was subjected to a heat shock for 40 seconds at 42 ℃and left for 2 minutes on ice. Then, 800µL of LB medium were added and the cells were shaken at 37℃ for 1h. Afterwards, the cells were centrifuged for 2 minutes at 2000 rpm, most of the supernatant was removed, leaving ∼ 200 µL. The cells were resuspended and added to to LB-Agar plates (LB-Agar 40 g dis-solved in 1 L H₂O) containing 100µg/mL ampicillin, 50 µg/mL kanamycin or 50µg/mL spectinomycin, according to the antibiotic resistance cassette incorporated to the vector of the mixture.The plates were incubated at 37℃for 16-18 h. For plasmid purification, E.

coli samples of colonies grown in antibiotic-containing plates were picked with autoclaved pipette tips, added to appropriate antibiotic-containing LB media (25 g LB-Medium per 1 L H₂O) and grown at 37 ℃for 16-18 h.

2.2.1.6 Plasmid DNA amplification and purification

Cultures of transformed DH5_alpha E. coli (Subcloning Efficiency DH5_alpha, Invitrogen) were grown in antibiotic-containing LB media H₂O) with constant shaking. The cells

1molar ratio

were centrifugated at 4 000gfor 15 min at 4℃and DNA purification was performed using NucleoBond Xtra Midi EF Kit (Macherey-Nagel) following manufacturer’s instructions.

Eluted DNA was reconstituted in sterile H₂O.

To check ligation effectivity by restriction enzyme digestion and sequencing, E. coli sam-ples of colonies grown in antibiotic-containing plates were picked with autoclaved pipette tips and grown in 3 mL LB containing the appropriate antibiotic for selection and grown at 37℃for 16-18 h with constant shaking. Plasmid DNA was purified from these cultures using NucleoSpin Plasmid QuickPure Kit (Macherey-Nagel) according to manufacturer’s recommendations.

2.2.2 Cell culture

2.2.2.1 Primary cultures

Primary oligodendrocyte cultures were prepared from the brain of postnatal day 0-2 rats or mice as described previously (Trajkovic et al., 2006). After removing the meninges, the neonatal brain hemispheres were digested with 0.25% trypsin,and cultured in Eagle

’s basal medium with 10% horse serum on poly-L-lysine (PLL)-coated flasks at 37 °C.

Oligodendroglial cells were harvested from 8-10 day old mixed glia culture using mechan-ical dissociation. Cells were cultured on PLL-coated dishes or glass coverslips in Super SATO medium (DMEM containing 1% horse serum, B27 supplement, L-thyroxine, tri-iodothyronine, glutamine, pyruvate, and penicillin/streptomycin, see materials section for protocol).

To obtain primary neuronal cells, mixed brain culture from embryonic day 16 mice was prepared as previously described (Fitzner et al., 2006). Cells were cultured in Super SATO medium for two weeks to acquire confluent differentiated neurons.

To prepare a coculture of neurons and oligodendrocytes, neurons were cultured in a 12-well plate for 2 weeks as described above. The media was exchanged for fresh Super SATO media and 60 000 oligodendrocyte precursor cells were added per well and allowed to grow for 5 days.

2.2.2.2 Cell line culture

Human Embryonic Kidney (HEK) 293T cells (Graham et al., 1977) were used for pro-duction of Fc-fusion soluble proteins and binding assays. The cells were cultured in high glucose DMEM, 1 × GlutaMAX, 10% FCS and 100 U/mL penicillin and streptomycin.

The cells were split 2-3 times per week after trypsinization with 0.05% trypsin/EDTA (Lonza GmbH). For Fc-fusion protein production, the media was exchanged for 1% FCS media.

2.2.2.3 Mammalian cells transfection and RNAi

Transfection of primary cultures and HEK cells was done using Lipofectamine® 2000 (Invitrogen). For cells cultured in 12 well plates, the following reaction was prepared per well: Per each coverslip in 12-well plates 1.6 µg of plasmid DNA was mixed with 100 with 100µL OptiMEM-I (Invitrogen). Separately, 4µL Lipofectamine2000 reagent were mixed with 100 µL OptiMEM. After 5 min incubation, both solutions were mixed and incubated for 20 min at RT. The 200µL solution was added dropwise to the cells and left at least overnight before analysis.

Knockdown of exogenously expressed Lsamp, Opcml and Ntm (IgLON family) in HEK 293T cells was done in 6-well dishes by mixing 250µL OptiMEM with 0.5µg of myc-tagged IgLON protein and 3 µg shRNA plasmid (see Materials section for details). 250 µL of OptiMEM was mixed with 10µL Lipofectamine 2000 in a separate vial and incubated for 5 min at RT. The two solutions were mixed gently, incubated for 20 min at RT and added dropwise to HEK 293T cells grown on PLL-coated 6-well dishes. After 3 days, the cells were scraped and prepared for analysis by Western blot.

2.2.2.4 Preparation of cell lysates for Western blot

To prepare cell lysates for analysis by Western blot, the culture medium was removed and the cells were washed once with PBS. An appropriate volume of Lysis buffer (2% NP-40, 0.2% SDS, 1 mM EGTA in PBS supplied with protease inhibitors) was added and the cells were detached using a cell scraper (Sarstedt), while being kept on ice. The samples were then centrifuged at 10 000 g for 10 min and the supernatant was used for protein concentration measurement using Bradford assay (Bradford, 1976). The samples were mixed with equal volume of 2 ×sample buffer (20% glycerol, 4 mM EDTA, 4% SDS, 4%

2-mercaptoethanol, 0.05% Bromophenol blue and 100 mM Tris-HCl pH 6.8) and stored at -20℃ until further analysis.

HEK cells were transfected with Calcium Phosphate method based on the formation of a calcium phosphate-DNA precipitate which is uptaken by cells by endocytosis (Wigler et al., 1977; Abrahams and Van der Eb, 1975). For soluble Fc-fusion production, cells were cultured in 20 cm culture dishes in 25 mL media per dish. A typical transfection reaction for one dish was prepared as follows:

Transfection reaction

Soluble Fc-fusion proteins were purified using Protein A HP Spin Trap columns (GE Healthcare), following manufacturer’s instructions. Briefly, HEK cells were transfected as described in the previous section. After 2-3 days, the supernatant was collected, and centrifuged for 15 min at 4 000 g at 4℃. 1×Complete Protease Inhibitor Cocktail (Roche) was added to the supernatant and then concentrated using Amicon Ultra-15 Centrifugal Filter Units (Millipore) according to manufacturer’s recommendations. The final volume of 2 mL was diluted in equal volume of binding buffer (20 mM sodium phosphate, pH 7.0).

Briefly, the storage solution from the column was removed by centrifugation for 30 s at 100 g. The column was equilibrated adding 600 µL of binding buffer, centrifuged for 30 s at 100 g. Then, 600 µL of the antibody solution were added, incubated for 4 min while gently mixing and then centrifuged for 30 s at 100 g. This procedure was repeated until all the volume was loaded on the column. Then the column was washed twice adding 600 µL binding buffer and centrifuging for 30 s at 100 g. Two collection tubes per sample were prepared for eluted fractions, each one containing 30µL of neutralizing buffer . The proteins were eluted twice by adding 400µL of elution buffer (0.1 M glycine-HCl, pH 2.7),

mixing by inversion, placing the column in a 2 mL microcentrifuge tube containing 30µL neutralizing buffer (1 M Tris-HCl, pH 9.0) and centrifuged for 1 min at 50 g. The proteins were aliquoted and kept at -20℃ .

2.2.4 Myelin isolation and purification

We isolated different fractions of myelin from C57BL6 mice (1-6) or human sample (7):

1. Standard compact myelin preparation in discontinous sucrose gradient, 0.32M/0.85M interphase.

2. Myelin isolated from postnatal day 14 (P14) brain homogenate, in discontinuous sucrose gradient, 0.32M/0.85M interphase.

3. Purified myelin solubilised with 1% TritonX overnight and bound to ConcanavalinA beads

4. Purified myelin bound to WGA beads.

5. Myelin isolated from brain homogenate subjected to an initial hyposmotic shock with water and then subjected to a continuous gradient, fraction 0.9-1M sucrose.

6. Myelin isolated from brain homogenate subjected to an initial discontinuous gradi-ents and collected from 0.32-1.2M interphase and subsequently subjected to a continuous sucrose gradient. Collected from Fraction 3, or 0.9-1 M sucrose.

7. Glycoprotein from human myelin preparation, subjected to a lentil-lectin column (Mathey et al., 2007), was kindly provided by Edgar Meinl (Max Planck Institute of Neurobiology, Munich).

2.2.4.1 Myelin isolation by centrifugation in discontinous sucrose gradient Compact myelin from 3-months-old mice (sample 1, 3 and 4) and 14-days-old mice (sample 2) were isolated based on a protocol previously described (Norton and Poduslo, 1973), with slight modifications. All the steps were done on ice using a SW41 Ti rotor in an Optima XL-70 ultracentrifuge (Beckman Coulter, USA). Homogenized brain tissue was placed over a discontinuous sucrose gradient (0.32 M and 0.85 M sucrose for fractions in a preparation buffer containing 5 mM EDTA and 10 mM Hepes, pH 7.4) and centrifuged for 30 minutes at 75 000 g. The interface between the two sucrose gradients was recovered and diluted in ten volumes of ice-cold water, and centrifuged for 20 minutes at 75 000 g.

The pellet was subjected twice to hypo-osmotic shock by resuspension in ice-cold water and 10 minutes centrifugation steps at 12 000 g. The pellet was resuspended on 0.32 M sucrose in preparation buffer and overlaid on 0.85 M sucrose (in preparation buffer) to

create a new gradient as the one mentioned above. The previous centrifugation steps were repeated and the final purified myelin pellet was resuspended in preparation buffer and kept at -20℃ for futher use.

2.2.4.2 Myelin isolation by centrifugation in continous sucrose gradient A continous sucrose gradient was prepared as follows: Using a long needle, 1 mL of 0.6 M sucrose in preparation buffer (5 mM EDTA and 10 mM Hepes pH 7.4) was laid in the bottom of a ultracentrifuge tube (Thinwall, Ultra-Clear™, 344059, Beckmann Coulter).

Then, successive 1.5 mL layers of sucrose with increasing molarity (0.7, 0.8, 0.9, 1.0, 1.1 and 1.2 M) were added. The tube was left for 3 hours at room temperature to allow the formation of a continous gradient. Then, samples 5 and 6 were prepared by overlaying either brain homogenate (sample 5) or myelin from a 0.32 /1.2 M sucrose interphase (sample 6) onto the continuous sucrose gradient described above. The samples were centrifuged at 75 000 g for 1 hour using a SW41 Ti rotor in an Optima XL-70 ultracentrifuge (Beckman Coulter, USA). The overlay remains and the first mL of the continous gradient were removed. Afterwards, 6 fractions of 1.5 mL each were taken, diluted in ice cold water and centrifuged for 1 hour at 75 000 g. Subsequently, the pellet was subjected twice to hypo-osmotic shock by resuspension in ice-cold water and 10 minutes centrifugation steps at 12 000 g, resuspended in preparation buffer and kept at -20℃ for further use.

2.2.4.3 Glycoprotein enrichment by WGA and ConA columns

To isolate glycoprotein-rich myelin fractions using Wheat Germ Agglutinin (WGA) lectin resin, purified myelin from adult mice was obtained as stated in section 2.1.1. The myelin suspension was passed through a 26G gauge needle 10 times and bound to a Wheat Germ Agglutinin (WGA) column (Pierce Glycoprotein Isolation Kit, WGA, Thermofisher) fol-lowing manufacturer’s instructions. After washing, the beads were retrieved, resuspended in sample buffer and boiled for 10 minutes at 70℃. The supernatant was retrieved and kept at -20℃for further use. To isolate myelin glycoproteins using Concanavalin A lectin resin, purified myelin from adult mice was initially obtained as stated in section 2.1.1.

The myelin was spun down and resuspended in a solution of 0.1% Triton X in PBS for solubilization. The supernatant was then loaded onto a ConA column (Pierce Glycopro-tein Isolation Kit, ConA, Thermofisher) according to manufacturer’s instructions. After

washing, the ConA beads were retrieved, resuspended in sample buffer and boiled for 10 minutes at 70℃ .

2.2.4.4 Human myelin glycoprotein enrichment

Glycoproteins were isolated from human myelin preparation with lentil-lectin affinity chro-matography according to the manufacturer’s guidelines (GE Healthcare) as described pre-viously (Mathey et al., 2007).

2.2.5 SDS-PAGE and Western Blotting

Protein analysis by Western Blot was performed by SDS-PAGE as described earlier (Bur-nette, 1981; Laemmli, 1970). The polyacrylamide gels comprising of stacking and resolving gels were self-cast with composition described in Table 2.11. Equal amounts of protein from each sample were mixed in a 1:1 volume proportion with 2X loading buffer (10%

glycerol, 2 mM EDTA, 2% SDS, 144 mM ß-mercaptoethanol, 50 mM Tris-HCl and 0.02%

bromophenol blue), heated for 10 min at 70℃ and loaded onto 12% SDS-PAGE gel. For each gel, 3.5 µL of pre-stained protein ladder was loaded into one of the lanes. Elec-trophoresis was performed at 100V in the running buffer (190 mM glycine, 25 mM Tris and 0.1% SDS). The proteins were transferred from the polyacrylamide gel onto a Protan nitrocellulose membrane (Whatman GmbH, Germany) using a semi-dry procedure, for 1 hr at 100 V in the transfer buffer (20 mM Tris base, 153 mM glycine, 20% methanol).

Table 2.11:Components for Western Blot gels

Stacking gel Resolving gel

H2O 1.21 mL H2O 1.66 mL

30% Acrylamide solution 0.27 mL 30% Acrylamide solution 2.04 mL

Stacking buffer (0.5 M Tris-HCl, pH 6.8) 0.5 mL Resolving buffer (1.5 M Tris-HCl, pH 8.8) 1.3 mL

10% SDS 20µL 10% SDS 50µL

10% APS 20µL 10% APS 50µL

TEMED 3µL TEMED 2µL

The correct transfer of protein was confirmed by fast reversible staining of protein bands by immersing the membrane in 0.1% Ponceau S in 5% acetic acid solution for five min-utes and thereafter washing it with distilled water. The blot was blocked with 4% milk powder in PBS for 1 hr at RT followed by overnight incubation with appropriate

pri-mary antibody diluted in PBS containing 0.1% Tween-20 (PBST). After washing three times for 10 minutes with 0.1% PBST, the membranes were incubated with the appro-priate horseradish peroxidase-conjugated secondary antibody diluted in PBST for 1 hour at room temperature. The blots were washed three times for 10 minutes with PBST and subjected to a mixture of equal parts of peroxide solution and luminol enhancer solution detection reagents (Thermo Scientific, Pierce). The blots were later exposed to light sensi-tive CL-XPosure films (Thermoscientific) and developed in a Kodak X-OMAT 1000 image processor. Images of the films were prepared using Adobe Photoshop software (Adobe Systems, Mountain View, CA).

2.2.5.1 Protein concentration determination

The protein concentration of cell lysate preparations, myelin preparations and purified proteins was determined by using Bradford assay (Bradford, 1976), and BCA Protein Assay (Pierce, Thermo Scientific) following manufacturer’s instructions. The absorbance at 562 nm of the mixtures was measured with a 96-well plate MRXTc Revelation reader (Dynex Technologies).

2.2.6 In vitro assays

2.2.6.1 Immunocytochemistry

For immunolabeling, cells were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature before permeabilization with 0.1% Triton X-100 in PBS for 1 min. The blocking solution (2% BSA, 0.2% Fish gelatine, and 2% FCS in PBS) was then added to fixed cells for 30 min at RT. The primary antibodies diluted in blocking solution were added for 1 h at RT or overnight at 4℃ . After washing three times with PBS, cells were incubated with secondary antibodies for 1 h at RT. Finally, the coverslips were washed with PBS and mounted onto a glassslide with a drop of Mowiol solution (2.4 g Mowiol, 6 g Glycerol, 6 mL H2O, 12 mL 0.2 M Tris/HCl pH 8.5) and were kept in at 4℃ in the dark. When required, nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI).

2.2.6.2 Paraformaldehyde (PFA) solution for fixation

Paraformaldehyde (PFA) 16% stock solution was prepared by dissolving 16 g PFA in 70 mL H2O, heating at 60℃ adding NaOH pellets until the solution became clear. Then, 10 mL 10 × PBS were added and pH was adjusted to 7.4. Water was added to a total

volume of 100 mL. Aliquots of the solution were then stored at -20℃ . Working solution of 4% PFA in PBS was prepared by diluting 50 mL of PFA stock solution in 150 mL of 1×PBS and stored at 4 ℃ .

2.2.6.3 Mowiol solution for immunocytochemistry

Mowiol was used as the mounting medium after immunocytochemistry. The solution was prepared in the following way: 2.4 g Mowiol was added to 6 g glycerol and 6 mL H2O.

The mixture was stirred for seveeral hours at room temperature. Then, 12 mL 0.2 M Tris-HCl (pH 8.5) was added and then incubated at 60 ℃ for 10 min. The solution was centrifugated at 4000 g for 15 min, aliquoted an kept at -20 ℃ until further use.

2.2.6.4 Binding assay

For binding assays, supernatant from transfected HEK cells were retrieved and centrifuged 10 min at 5000 rpm. Per each 18 mm coverslip of neuronal or oligodendroglial culture, 150 µL of media were mixed with 1.5µL of Cy3-conjugated anti-human Fc antibody (Dianova) and incubated for 30 min at RT. Then, the mix was added to each coverslip and incubated for 20 min at RT in a humid chamber (Feinmechanik Service Department, Max Planck Institute for experimental medicine). Finally, the coverslips were washed 3 times with PBS and the cells were fixed with 4% PFA for 15 min at RT.

2.2.6.5 Proliferation assay

For proliferation assays, 30 000 oligodendrocyte precursor cells (OPC) per well were plated in a 24-well plate on PLL-coated coverslips. 10µg/mL of the Fc-fusion proteins were then added to the wells. The cells were allowed to settle down and were fixed after 8 hours, and stained for the OPC marker A2B5. We used 100 ng/mL PDGF as a positive control of proliferation.

2.2.6.6 Migration assay

Migration assays were performed using CytoSelect 24-Well Cell Migration and Invasion Assay (8 µm, Colorimetric Format). Briefly, the chamber membranes were coated by placing each insert on 400µL PLL (100µg/mL) for 1 h at 37℃ and washed 2 times with PBS. A cell suspension of 1 000 000 oligodendrocyte precursor cells per mL was prepared in serum-free Super SATO. 500µL of serum-free super SATO media containing 10µg/mL

Fc-fusion proteins, 100 ng/mL PDGF or equal volume PBS were added to the lower well of the migration plate. Next, 300µL of OPC suspension were added to the inside of each insert, and incubated 8 hours at 37℃ . Then, the media from the inside of the insert was removed, cotton-tipped swabs were used to remove the interior of the inserts to remove non-migratory cells. The insert was transferred to a clean well containing 400 µL of Cell Stain Solution and incubated for 10 minutes at room temperature. After allowing the inserts to air dry, migratory cells were counted with a Nikon Ti-E brightfield microscope.

2.2.6.7 Differentiation assay

To observe whether the presence of soluble IgLON in the media can influence the growth and differentiation of oligodendrocytesin vitro, OPC were grown for 4 days in media with 30 µg/mL of IgLON Fc-fusion proteins on PLL-coated coverslips, and then fixed with 4%

PFA.

2.2.6.8 Adhesion assay

Glass coverslips in a 24-well plate were coated with 500µL of a solution with 10µg/mL of donkey anti-human Fc antibody (Dianova) in 50 mM Tris-HCl (pH 9.0) and left overnight at 4℃. Then, the coverslips were washed 3 times with supplement-free DMEM and 500 µL of a solution containing 10 µg/mL of Fc-fusion protein in 0.2% BSA/PBS. After 1h of incubation at 37℃ , the coverslips were washed 3 times with DMEM and fresh Super SATO media was added. PLL coating (100µg/mL was used as a positive control to verify the quality of the primary oligodendrocyte preparation. After washing and placing new media, 25 000 OPC were plated and allowed to grow for four days, and fixed with 4%

Glass coverslips in a 24-well plate were coated with 500µL of a solution with 10µg/mL of donkey anti-human Fc antibody (Dianova) in 50 mM Tris-HCl (pH 9.0) and left overnight at 4℃. Then, the coverslips were washed 3 times with supplement-free DMEM and 500 µL of a solution containing 10 µg/mL of Fc-fusion protein in 0.2% BSA/PBS. After 1h of incubation at 37℃ , the coverslips were washed 3 times with DMEM and fresh Super SATO media was added. PLL coating (100µg/mL was used as a positive control to verify the quality of the primary oligodendrocyte preparation. After washing and placing new media, 25 000 OPC were plated and allowed to grow for four days, and fixed with 4%

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