Animals and reagents
A colony of translin knockout mice was established at Johns Hopkins from the line generated in Dr. M. Kasai’s laboratory (Fukuda et al., 2008
). These mice had been backcrossed to C57/BL6 for over 10 generations. The translin knockout mice (Nbio055) were provided by the JCRB Laboratory Animal Resource Bank of the National Institute of Biomedical Innovation (Osaka, Japan). Genotyping of mice was performed on DNA isolated from tail snips. PCR was conducted using the primers and conditions described by Fukuda et al. (2008)
. Animal housing and procedures adhered to IACUC guidelines.
Rabbit polyclonal translin antiserum had been generated against a fusion protein containing the full-length rat translin sequence fused to glutathione-S-transferase (Finkenstadt et al. 2000
). Two trax antibodies were used: one is a rabbit polyclonal antibody provided by Dr. Chern’s laboratory (Sun et al., 2006
); the other is a guinea pig polyclonal antibody generated previously (Finkenstadt et al. 2000
; Finkenstadt et al. 2001
A myc-His-translin construct was generated as described in Finkenstadt et al. (2002)
except that it encodes the human translin orthologue. An mCherry tagged trax construct was generated by inserting the native rat trax coding region into the HindIII/KpnI restriction sites of Clontech’s pEGFP-C1 vector in which the eGFP region had been replaced with mCherry (gift from M. Meffert). The mCherry tagged Translin construct was also cloned by inserting the rat translin coding sequence into the aforementioned vector using HindIII and SacII restriction sites. Translin GFP and trax GFP constructs were generated by inserting their respective rat coding sequences into the XhoI/EcoRI cloning sites in pEGFP-N1 (Clontech).
Primary Hippocampal Cultures
For immunohistochemical studies, rat and mouse hippocampal cultures were prepared from E18 embryos that were dissected quickly on ice in the following medium: Hank’s balanced salt solution supplemented with 100 U/ml penicillin and 100 U/ml streptomycin (pen/strep: Invitrogen), 1 mM MEM sodium pyruvate (Invitrogen), 30 mM glucose and 10 mM HEPES (Invitrogen). Tissue was then digested with papain (0.67mg/ml; Worthington) and 1% DNase (Sigma) at 37°C for 20 minutes. Following digestion, tissue was rinsed three times followed by trituration in neurobasal medium (Invitrogen) supplemented with 5% fetal bovine serum (FBS) (Hyclone, Logan, UT, USA), 2 mM Gluta-MAX-I (Invitrogen), and 100U/ml pen/strep. The resulting cell suspension was passed through a 70 μm filter. Cells were then plated on polylysine-coated 12 mm coverglasses at a density of 3×104 cells (rat) or 2×105 cells (mouse) in 24 well culture dishes. Neurons were fed glia-conditioned, 1% FBS neuronal medium supplemented with 2% B27 (Invitrogen) every 3–4 days. Neurons were transfected using Lipofectamine 2000 (LF2000; Invitrogen) following manufacturer’s directions.
To monitor BDNF mRNA trafficking in vitro, primary hippocampal neurons were prepared from P2 rats as described (Tongiorgi et al., 1997
Forebrains were collected from 3 month old mice and immediately homogenized in cold lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 4 mM EDTA, 1% (v/v) NP-40 and 2X Complete mini EDTA-free protease inhibitor complex (Roche Molecular Biochemicals, Indianapolis, IN, USA). Forebrain homogenates were incubated on ice for 10 minutes then centrifuged at 15000 g for 10 minutes at 4°C. The supernatant was then harvested and Laemmli sample buffer added prior to heating the samples for 5 minutes in a 100°C water bath. Proteins were separated by electrophoresis on 10% polyacrylamide gels and then transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were probed with polyclonal trax antibody (guinea pig: 1:500), polyclonal translin antibody (rabbit: 1:10,000) and mouse monoclonal anti-β-Tubulin type III antibody (T8860 Sigma, St Louis, MO, USA: 1:500) in 1% non-fat dry milk and 0.05% Tween-20 in Tris-buffered saline. After washing the membrane, horseradish peroxidase-conjugated secondary antibodies (anti-rabbit and anti-mouse; Amersham Pharmacia Biotech AB, Piscataway, NJ, USA; goat anti-guinea pig, Chemicon) were used at 1:5000. The bands were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech AB).
Adult mouse forebrain was collected and immediately homogenized in 1 ml of cold lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.2% deoxycholate and 2X Complete mini EDTA-free protease inhibitor complex (Roche Molecular Biochemicals, Indianapolis, IN, USA)). Homogenates were kept on ice for 20 minutes then centrifuged at 15000 g for 10 minutes at 4°C. Supernatant was then harvested and pre-cleared with protein A agarose beads (Pierce) for 30 minutes at 4°C. Supernatant was then collected after a 5 minute spin at 5000 g and 30 μl was kept aside as “offered”. One half of the remaining volume (~485 μl) was mixed with 10 μl of the guinea pig polyclonal trax antibody, while the other half was processed further in the absence of trax antibody. Both tubes were rocked at 4°C for 1.5 hours. Protein A agarose beads were then added to both tubes which were allowed to rock at 4°C for another hour. Beads were then spun down at 5000 g and supernatants removed. Fifty μl of the supernatant from the tube containing trax antibody was kept and designated as “S+”. Beads were washed three times with 500 μl lysis buffer for 5 minutes each at 4°C. After the last wash, 50 μl of 2X Laemmli buffer were added to the beads and S+ sample and 30 μl of 2x Laemmli to the offered sample. All samples were boiled for 5 minutes and then stored at -20°C. Proteins were separated by electrophoresis on 10% polyacrylamide gels and then transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were probed with polyclonal trax antibody (guinea pig: 1:500) and polyclonal translin antibody (rabbit: 1:10,000) as described above.
Electrophoretic Mobility Shift Assays
Gel-shift assays were performed as described previously (Li et al. 2004
). In brief, freshly dissected cerebella and hippocampi from translin wt and ko mice were homogenized in a solution containing 20 mM HEPES (pH 7.9), 400 mM NaCl, 20% glycerol, 1.5 mM MgCl2
, 20 mM NaF and protease inhibitor cocktail (Roche), incubated on ice for 15 min, and then centrifuged at 15,000×g
at 4°C for 15 min. Supernatants were stored at −80 °C for later use.
Radiolabeled probe was prepared by incubating a 39-mer RNA oligo sequence which corresponds to a segment of the 3’UTR of protamine-2 (Li and Baraban, 2004
) with [γ-32
P] ATP and T4 polynucleotide kinase (New England Biolabs) and purified with a Sephadex G-50 column (GE Healthcare). Five μg of extract protein was incubated with 20,000 cpm of probe in 12 mM HEPES (pH 7.9), 4 mM Tris–HCl (pH 7.9), 50 mM KCl, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 12% glycerol, and 2 μg of poly(dl–dC) in 30 μl total reaction volume for 15 min at room temperature. The reaction was loaded onto a 5% native polyacrylamide gel. After electrophoresis, gels were dried and exposed to Biomax-MR (Kodak) film overnight at −80 °C.
For immunostaining studies of endogenous translin and trax in brain sections, mice were anesthetized with chloral hydrate (400 mg/kg, i.p.) and then perfused via cardiac puncture with chilled PBS (1 mM KH2P04, 10 mM Na2HP04, 137 mM NaCl, 2.7 mM KCl, pH 7.4), followed by freshly prepared 4% paraformaldehyde (in PBS). Brains were post-fixed in this solution overnight and then cryoprotected by immersion for at least 24 hours in 25% sucrose dissolved in PBS. Thirty μm sections were cut on a sliding microtome. For trax immunostaining, blocking and tissue permeabilization were achieved by incubation of sections in 30mg/ml BSA and 0.1% Triton-X-100 in PBS for one hour. Sections were then incubated overnight at 4°C with polyclonal anti-Trax (rabbit 1:5000) diluted in PBS with 10mg/ml BSA. Extensive washing in PBS was followed by incubation with biotinylated anti-rabbit (1:2000; Vector Labs) overnight at 4°C. The brain sections were then washed in PBS and incubated for 30 minutes with ABC (Vectastain Kit: Vector Labs), and developed for 10 minutes with the tyramide signal amplification solution (1:400; TSA Plus fluorescein, Perkin Elmer). A brief wash period with PBS was followed by 10 minute incubation with DAPI prior to mounting and coverslipping with Permafluor-DABCO (Beckman-Coulter, Marseille, France).
For translin immunostaining, sections were processed in a similar fashion except that an antigen retrieval step was included after collecting the sections in PBS. In this protocol, sections were processed as described below for the in situ hybridization procedure up to primary antibody step, which included an overnight incubation at 60°C. After washing the sections with PBS, they were incubated overnight in polyclonal rabbit translin antibody (1:6000) and the staining procedure completed as described above for trax staining. As the inclusion or omission of the antigen retrieval step abolished trax and translin staining in brain sections, respectively, double staining could not be performed on brain tissue.
For co-localization studies of recombinant mCherry Translin and Trax GFP in mouse cultures, neurons were transfected with 100ng of each construct at 7 days in vitro (DIV). The following day (18-24 hours later), neurons were rinsed briefly in PBS and then fixed in 4% formaldehyde (in PBS) for 15 minutes. They were then permeabilized in PBS containing 0.1% Triton X-100 for 10 minutes followed by an hour blocking step in 50 mg/ml bovine serum albumin (BSA) in PBS. Cultures were then incubated overnight with mouse anti-MAP2 antibody (1:250, Chemicon International), diluted in PBS with 10mg/ml BSA. After several washes in PBS the following day, the coverslips were incubated for 60 min at 25°C with anti-mouse Alexa-fluor 405 (1:500, Invitrogen) diluted in PBS with 10mg/ml BSA. After several washes in PBS, coverslips were mounted onto slides with Permafluor-DABCO (Beckman-Coulter, Marseille, France). In rat cultures where we stained for both endogenous trax and recombinant translin, neurons were transfected at 9 DIV with a myc-His-tagged translin construct (100 ng) and then processed for staining 18-24 hours later. In this case, cells were incubated simultaneously with both the rabbit trax antibody and a mouse monoclonal anti-myc antibody (1:5000; Invitrogen), followed by incubation with a mixture of anti-rabbit Alexa-fluor 555 and anti-mouse Alexa-fluor 488 (each at 1:2000, Molecular Probes).
The specificity of the polyclonal Trax antibody in rat cultures was determined via co-transfection of rat hippocampal neurons with 100ng eGFP plasmid with siRNA oligos (Dharmacon) at 6 DIV using 30, 50 or 100 nM concentrations. siRNA oligos targeting the following sequences in rat Trax were used: 835: AAGUGGAGAACGCUUGCUA and 218: GGACACAAGACACGACAAA. As control, we also tested a mutant form (835M) of the 835 siRNA with the following sequence: CAGUGGCGAACGAUGAUA (bold indicates nucleotide substitutions). Three days post-transfection, cultures were fixed and processed for staining as described above, using the rabbit Trax polyclonal antibody (1:5000) and the anti-rabbit Alexa-fluor 555 secondary antibody.
Mice treated with pilocarpine were 2-3 months old. To reduce mortality from peripheral cholinergic effects, mice were pre-treated with methylscopolamine (1 mg/kg, i.p.,Sigma) dissolved in PBS 20 minutes before receiving pilocarpine (250 mg/kg, i.p., Sigma) dissolved in 0.9% NaCl (Turski et al., 1989
). Only mice which developed generalized seizure activity within an hour of pilocarpine administration were used for histological studies. At three hours following pilocarpine administration, mice were anaesthetized with chloral hydrate (400 mg/kg, i.p., Sigma) dissolved in PBS and perfused via intracardiac puncture with freshly made 4% paraformaldehyde (PFA) solution made in DEPC-treated PBS. Control mice were only treated with chloral hydrate prior to perfusion. The brains were then removed and placed overnight in 4% PFA prepared DEPC-treated PBS at 4°C. The next day, brains were transferred to a solution consisting of 25% (w/v) sucrose in DEPC-treated PBS. On the following day, 30-35 μm hippocampal sections were cut on a sliding microtome and collected in DEPC-treated PBS. Sections were then mounted on Superfrost Plus slides (VWR) and processed for in situ
In situ hybridization
To monitor the localization of BDNF mRNA in hippocampal sections, hybridization was performed with an antisense probe targeting nucleotides 240-666 of exon IX (NM_007540). The corresponding sense probe was used as a control. To generate these probes, total RNA was isolated from mouse hippocampus and cDNA obtained by reverse transcription. PCR primers targeting this segment contained 5’ HindIII and 3’ XbaI overhangs and the PCR product was cloned into PCR3.1 (Invitrogen). The following primer sequences were used: fwd - 5’CCCAAGCTTGCTGGATGAGGACCAGAAGGT3’ and rev -5’GCTCTAGAGCTTGGGTAGTTCGGCATTGCGA3’. Digoxigenin-labeled RNA probes were generated by using the DIG RNA labeling mix (Roche Applied Sciences) and T7 polymerase. Sense probe was generated by reversing the 5 and 3’ restriction site overhangs and inserting the PCR product in reverse into the plasmid and then continuing with T7 mediated transcription.
Brain sections were post-fixed in 4% paraformaldehyde (PFA) followed by washes in PBS and then permeabilized in PBS with 0.3% Triton-X100. Sections were then rinsed in PBS and treated with proteinase K (1 μg/ml) at 37°C in buffer (100 mM Tris, 50 mM EDTA, pH 8) and treated with acetic anhydride (in 0.1M TEA, pH 8). Slides were washed in PBS and blocked in pre-hybridization buffer (50% deionized formamide, 5X SSC, 1X Denhardt’s, 250 μg/ml yeast tRNA) at 60°C and then hybridized with DIG labeled probe in hybridization buffer (same as pre-hybridization buffer but with 10 mM DTT and 0.5 mg/ml salmon sperm). On the next day, slides were washed sequentially at 60°C in 50:50 2X SSCT (SSC and 0.1% triton-X100): formamide, 2X SSCT, and 0.2X SSCT. Sections were then washed in TBS-T (100 mM Tris, 150 mM NaCl, pH 7.5, 0.1% Tween-20) and blocked in TBS-T containing 5% BSA at room temperature. Sections were then incubated overnight with anti-DIG antibodies that were coupled to peroxidase (1:500; Roche Applied Science) in 1% BSA in TBS-T. On the third day, sections were washed in TBS-T and developed for 10 minutes using the TSA Plus fluorescein kit (1:100; Perkin Elmer). Sections were then washed in PBS and coverslipped.
To monitor BDNF mRNA localization in hippocampal cultures, cultures were treated with an RNAi “cocktail” against translin generated as described (Chiaruttini et al., 2009
) on DIV 7, and then stimulated with BDNF (50 ng/ml) or KCl (10mM) the following day. After 3h of stimulation, cultures were fixed and processed for in situ hybridization with a probe targeting exon IX (Chiaruttini et al., 2009
To quantify the in situ hybridization signal on brain sections, images were acquired at 20x power using a Zeiss Axiocam MRM camera mounted on an AxioExaminer D1 microscope and then quantified using Image J software (NIH). To measure the intensity of labeling in the stratum radiatum of CA1, we determined the average intensity of staining in rectangles 50 μm wide and 15 μm high positioned 5 to 20 μm, 20 to 35 μm, 35 to 50 μm and 50 to 65 μm away from the cell body layer of CA1. Background measurements taken over adjacent white matter were subtracted from the intensity measurements obtained for cell body or dendritic regions. Dendritic intensity values were divided by cell body labeling intensity to obtain a dendritic trafficking index. Data obtained at each of the dendritic intervals from four experimental groups, wt control (n=5), wt pilocarpine, (n=8), ko control (n=6) and ko pilocarpine (n=8), were then analyzed for statistical significance using 2×2 ANOVA.
Images of BDNF mRNA localization in hippocampal neurons were acquired with a CCD camera and analyzed with Image-ProPlus (Media Cybernetics). The relative dendritic filling (RDF) value was determined as described (Chiaruttini et al., 2009
Immunocytochemical images of cultured neurons were captured at a resolution of 1024×1024 pixels using the Zeiss Axiovert 200 with 510-Meta confocal module using 63X Plan-apochromat/ 1.4 oil DIC objective and 405 diode laser, 488 argon laser and 542 green HeNe laser and the Zeiss LSM 510 software. Images of Translin and Trax immunostaining and BDNF mRNA in situ hybridization were captured at a resolution of 1024×1024 pixels using the 10X Plan-apochromat objective, the 20X/0.75 Plan-apochromat objective, and the 40X/1.3 Plan-Neofluar oil DIC objective.
To determine the specificity of the rabbit polyclonal Trax antibody, we captured images at 20x power using a Zeiss Axiocam MRM camera mounted on an AxioExaminer D1 microscope, of the EGFP signal and endogenous Trax staining of 9 DIV rat hippocampal cultures at the same exposure across two different conditions: concentration (30, 50, 100 nM) and oligo target (835, 835M, 218). We measured the intensity of the cell body using Image J software (NIH) and normalized the Trax levels to the eGFP levels (took the ratio of the eGFP reading to 255 which is the maximum intensity and multiplied the Trax intensity by that ratio). We averaged all the values per group (n=12, 4 images at each concentration per oligo type).