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23 protocols using BDNF

Seven DIV neurons, plated at a density of 106 cells/cm2, were incubated in B27-free neurobasal medium for 1 h and then treated with 50 ng/mL of BDNF (Alomone). After washing, neurons were lysed in RIPA buffer (50 mM Tris, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 0.5% sodium deoxycholate, 1% NP-40, and 0.1% SDS) and supplemented with protease inhibitors cocktail (Roche) and phosphatase inhibitors cocktail (Roche, Basel, Switzerland). The homogenates were cleared by centrifugation at 14.000 rpm for 10 min. Protein extracts (50–70 µg) were loaded onto 10% SDS-PAGE and transferred to nitrocellulose membranes (Fisher Thermo Scientific). The membranes were blocked with 3% BSA in PBS for 1 h at room temperature, followed by overnight incubation at 4 °C with primary antibodies. After incubation with the appropriate HRP-conjugated secondary antibody (1:3000, Thermo Scientific), membranes were incubated with ECL substrate for detection of HRP enzyme activity (Thermo Fisher Scientific) and visualized in a Syngene gel documentation system. Images were quantified by ImageJ analysis (National Institutes of Health). 2–4 biological replicates were performed for every experiment. The number of repetitions for every experiment is stated in the corresponding figure description. The Student’s t-test was used for statistical significance assessment.
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TrkB inhibitor (ANA-12, N-[2-[[(Hexahydro-2-oxo-1H-azepin-3-yl)amino]carbonyl]phenyl]-benzo[b]thiophene-2-carboxamide Cat#SML0209) and 3-nitropropionic acid (3NP, Cat#N22908) 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT, Cat#M5655) were obtained from Sigma-Aldrich Argentina. Ultrapure bacterial lipopolysaccharide (LPS) (Escherichia coli, 0111:B4, Cat#tlrl-3pelps) was from Invivogen (USA); BDNF was purchased from Alomone (Cat#B-250). Culture media and supplements were acquired from Invitrogen Argentina. All other media and supplements were obtained from Sigma-Aldrich Argentina, unless otherwise specified.
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In vitro primary GC cultures were prepared from cerebella of PN6–PN7 wt and Npc1nmf164 littermates, as previously described [31 (link)].
A Boyden chamber cell migration assay was performed as described in [32 (link)]. Briefly, purified GCs in serum-free Dulbecco’s Modified Eagle Medium (DMEM) were placed in the poly-lysine precoated upper Boyden chamber while BDNF (40 ng/mL, #B-250, Alomone Labs, Jerusalem, Israel) was added to the lower chambers in the same medium. After overnight incubation at 37°C in 5% CO2, the upper surface of the membranes was scraped free of cells and debris. The membranes and covers were washed, fixed, and stained with Hoechst (Hoechst-33258, Invitrogen, Milan, Italy). Cells adhering to the underside of the membrane were counted under an epifluorescence Zeiss microscope with a 10× objective. The total number of cells that had migrated was calculated in five adjacent fields and the average values of each experimental group were expressed in arbitrary units, normalizing to the untreated control.
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The rats were anesthetized with isoflurane (5% isoflurane, 95% O2) and perfused with ice-cold sucrose artificial cerebrospinal fluid (aCSF) solution. From the rat brains, the hippocampus was dissected and samples were homogenized for 10 min with lysis buffer (1% Triton X-100, 0.32 M sucrose in HEPES solution) on ice with Halt protease/phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, CA, USA). Protein concentration was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, CA, USA). Equal amounts of protein were loaded onto a 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel for separation and then transferred onto 0.45-μm size pore polyvinylidene fluoride membranes. The membranes were blocked with 5% nonfat dry milk plus 0.1% Tween 20 for 2 h, and protein levels were analyzed by immunoblotting with primary antibodies, including GluN1, GluN2A, GluN2B (Sigma Aldrich, St. Louis, MO, USA), BDNF (Alomone, Jerusalem, Israel), and β-actin for normalization (SCBT, Dallas, TX, USA). All full blots are represented in Figs. S13, S14.
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Drugs were included in culture medium at DIV 9 for 3 days. BDNF (Alomone Labs) was used at the final concentration of 25, 50 ng/ml, in Phosphate-buffered saline solution (PBS). Mirtazapine (Abcam Biochemicals; Cat. N. ab120068) was tested at the concentration of 1, 5 or 10 µM (in DMSO 0.1%).
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For intracerebral infusions, 30-Gauge needles connected to Hamilton syringes were used. The volume infused was 0.5 μ l/side (LHb) and 1 μ l/side (mPFC and hippocampus) and the infusion rate was 0.25-0.5 μ l/min. Infusions were delivered through a needle extending 1 mm beyond the tip of the guide cannula. The needle was left in place for additional 120 s to minimize backflow. During the procedure, the animals were slightly restrained with the hands, without provoking any evident stress as mentioned in the previous section. Drugs and doses were as follow: anisomycin, 50 μg/side; emetine, 50 μg/side; muscimol, 30 ng/side; SKF 38393, 12.5 μg/side; human-BDNF (referred as BDNF), 0.5 μg/side. Drugs were dissolved in saline, except for SKF 38393 (saline 10% DMSO). Drugs were purchased from Sigma-Aldrich, except BDNF, purchased from Alomone labs.
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Primary SC neurons were cultured using E12.5 mouse embryos of either sex as previously described (Zahavi et al., 2015 (link)). Briefly, SCs were excised, trypsinized, and triturated. Supernatant was collected and centrifuged through a 4% BSA cushion. The pellet was resuspended and centrifuged through an OptiPrep gradient (10.4% OptiPrep, Sigma-Aldrich; 10 mm Tricine, 4% glucose) for 20 min at 760 × g with the brake turned off. Cells were collected from the interface, washed once in complete medium, and then plated in coated growth chambers. Cells were maintained in Complete Neurobasal Medium (Invitrogen) containing B27 (Invitrogen), 10% (v/v) horse serum (Biological Industries), 25 nm β-mercaptoethanol, 1% penicillin-streptomycin (PS; Biological Industries), and 1% GlutaMAX (Invitrogen) supplemented with 1 ng/ml GDNF, 0.5 ng/ml CNTF, and 1 ng/ml BDNF (Alomone Labs). Before plating, the growth plates were coated with 1.5 g/ml poly-dl-ornithine (Sigma-Aldrich) overnight at 37°C and 3 g/ml laminin (Sigma-Aldrich) for 2 h at 37°C. For immunofluorescence staining, 30,000 cells were plated on cover slides in 24-well plates. Cells were grown at 37°C in 5% CO2.
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Hippocampal tissues were homogenized on ice and lysed in lysis buffer containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.5% deoxycholic acid, 1% Nonidet P40, 0.1% sodium dodecyl sulfate, 1 mM PMSF, and leupeptin 100 mg/mL. The protein content was measured using a Colorimetric Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Thirty micrograms of protein was separated on sodium dodecyl sulfate-polyacrylamide gels and transferred onto a nitrocellulose membrane, which was incubated with mouse β-actin (1:1000; Santa Cruz Biotechnology, CA, USA), Bax (1:1000; Cell Signaling, Danvers, MA, USA), Bcl-2 (1:1000; Santa Cruz Biotechnology, CA, USA), BDNF (1:1000; Alomone, Jerusalem, Israel), PSD95, 1:1000; Cell Signaling, Danvers, MA, USA), primary antibodies. Horseradish peroxidase-conjugated secondary anti-mouse antibodies were used for β-actin and Bcl-2; anti-rabbit conjugated secondary antibodies were used for, Bax, BDNF, and PSD95.
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Neuronal induction of iPSCs was performed using a previously described method (Sato et al., 2021 (link)), with slight modifications. Briefly, iPSCs were seeded on iMatrix511-coated 12-well plates at a density of 1.0–1.5 × 105 cells/well in StemFit AK02 N medium. After 3 d, neural induction was initiated by changing the medium to neural induction medium [consisting of Advanced DMEM/F-12 (Thermo Fisher Scientific), 2% B27 supplement (–vitamin A; Thermo Fisher Scientific)] with 150 nm LDN193189 (StemRD), 5 μm SB431542 (Tocris), and 3 μm IWR1e (Calbiochem). On day 6, the cells were dissociated into single cells using Accutase (Nacalai) and seeded onto poly-L-ornithine-coated and laminin-coated 12-well plates at a 1:1–1:2 ratio. On day 12, the cells were dissociated again, and 8 × 105 cells seeded in each well of poly-L-ornithine-coated and laminin-coated six-well plates and cultured in neuronal medium [Advanced DMEM/F-12, 2% B27 supplement, 200 μm ascorbic acid (Sigma), and 200 μm dbcAMP (Sigma)] with 20 μm DAPT (Sigma). On day 18, DAPT was removed, and 10 ng/ml BDNF (Alomone Labs), 10 ng/ml GDNF (Alomone Labs), and 1 μm PD0332991 (Sigma) were added. The medium was changed every 3 d.
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Hippocampal neurons were plated at a density of 106 cells/cm2. At 7DIV neurons were incubated in B27-free neurobasal medium and then stimulated with BDNF 50 ng/mL (Alomone) for 1 h. Next, neurons were washed and lysed in immunoprecipitation assay buffer (20 mM Tris, 150 mM NaCl, 10% glycerol, 1% Igepal, 2 mM EDTA) supplemented with inhibitors (1 mM PMSF, 1 mg/mL aprotinin, 10 mg/mL leupeptin, 1 mM Na3VO4, and 50 mM NaF). Cell lysates were centrifuged at 14,000 rpm for 15 min at 4 °C. Protein quantification was performed using the Pierce® BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA). For immunoprecipitation assays, 300–500 µg of total lysates were incubated with 2 µg of anti-c-Abl (K12 Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-TrkB (Millipore) antibodies overnight at 4 °C. Complexes were isolated using protein G-Plus agarose (Santa Cruz cat# sc-2002). Immunocomplexes were subjected to SDS-PAGE and transferred to nitrocellulose membranes (Fisher Thermo Scientific) and analyzed by western blot using anti-TrkB (1:1000), anti-c-Abl (1:1000), and anti-GAPDH (1:5000) antibodies.
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