Brain derived neurotrophic factor (bdnf)

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BDNF is a recombinant protein that functions as a growth factor for various cell types. It is a member of the neurotrophin family and plays a critical role in the survival, growth, and differentiation of neurons.

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506 protocols using brain derived neurotrophic factor (bdnf)

Neuronal Differentiation Media Formulations

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N2B27 medium is composed of DMEM/F12 supplemented with modified N2 supplement and Neurobasal medium supplemented with B27 supplement minus vitamin A, in a 1:1 ratio and 50 μM β-mercaptoethanol (Thermo Scientific). FEB medium is N2B27 medium supplemented with 10 ng/mL FGF-2 (Peprotech), 10 ng/mL EGF (R&D Technologies), and 20 ng/mL BDNF (Peprotech). SFA medium is N2B27 medium supplemented with 100 ng/mL FGF-8 (Peprotech), 200 ng/mL sonic hedgehog (Peprotech), and 100 μM ascorbic acid 2-phosphate (Sigma). BGAA medium is N2B27 medium supplemented with 20 ng/mL BDNF, 10 ng/mL GDNF (Peprotech), 500 μM dibutyryl cyclic AMP (Sigma), 100 μM ascorbic acid 2-phosphate, 100 units/mL of penicillin, 100 μg/mL of streptomycin (Pen-Strep, Thermo Scientific), and 2 μg/mL Laminin (Roche).
Cyno NPC differentiation medium is composed of Neurobasal medium supplemented with B27 supplement (Thermo Scientific) and GlutaMAX supplement (Thermo Scientific), 20 ng/mL BDNF, 10 ng/mL GDNF, 100 μM ascorbic acid 2-phosphate, 100 units/mL of penicillin, 100 μg/mL of streptomycin, and 2 μg/mL Laminin.

Easton A., Jensen M.L., Wang C., Hagedorn P.H., Li Y., Weed M., Meredith J.E., Guss V., Jones K., Gill M., Krause C., Brown J.M., Hunihan L., Natale J., Fernandes A., Lu Y., Polino J., Bookbinder M., Cadelina G., Benitex Y., Sane R., Morrison J., Drexler D., Mercer S.E., Bon C., Pandya N.J., Jagasia R., Ou Yang T.H., Distler T., Grüninger F., Meldgaard M., Terrigno M., Macor J.E., Albright C.F., Loy J., Hoeg A.M., Olson R.E, & Cacace A.M. (2022). Identification and characterization of a MAPT-targeting locked nucleic acid antisense oligonucleotide therapeutic for tauopathies. Molecular Therapy. Nucleic Acids, 29, 625-642.

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Directed Differentiation of iPSCs to iNeurons

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The iPSCs were treated with 0.5 mM EDTA and plated as dissociated cells in E8 medium containing 5 μM Y-27632 (MedChemExpress, Monmouth Junction, NJ, USA) at a density of 10⁵ cells/mL on vitronectin-coated dishes on day −1. On day 0, the culture medium was replaced with N2/DMEM/F12/NEAA containing human brain-derived neurotrophic factor (BDNF, 10 mg/L, Peprotech, Inc., Rocky Hill, NJ, USA), human NT-3 (Neurotrophin-3, 10 mg/L, Peprotech, Inc., Rocky Hill, NJ, USA), mouse laminin (0.2 mg/L, Invitrogen Life Technologies, Carlsbad, CA, USA), and doxycycline (2 mg/L). On day 1, a 24 h puromycin selection (1 mg/L) period began. On day 2, transfected cells were replated in neurobasal medium supplemented with B27/Glutamax (Invitrogen Life Technologies, Carlsbad, CA, USA) containing BDNF and NT-3. On day 5, Ara-C (2 μM, Sigma-Aldrich, St. Louis, Missouri, USA) was added to the medium for 24 h to inhibit proliferation of undifferentiated cells. Subsequently, half the medium in each dish was changed every 3–4 days. Induced neurons were assayed on day 28 in most experiments.

Ko H.J., Chiou S.J., Wong Y.H., Wang Y.H., Lai Y.L., Chou C.H., Wang C., Loh J.K., Lieu A.S., Cheng J.T., Lin Y.T., Lu P.J., Fann M.J., Huang C.Y, & Hong Y.R. (2019). GSKIP-Mediated Anchoring Increases Phosphorylation of Tau by PKA but Not by GSK3beta via cAMP/PKA/GSKIP/GSK3/Tau Axis Signaling in Cerebrospinal Fluid and iPS Cells in Alzheimer Disease. Journal of Clinical Medicine, 8(10), 1751.

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BDNF Signaling in Granulosa Cells

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GCs were cultured in 6-well plates, with 10⁶ cells per well, until cell density reached 70% and then washed twice with PBS to eliminate the suspended cells. The culture media were replaced with serum-free DMEM/F12 for 4 h, and then cells were treated with BDNF, K252α (TrkB-specific inhibitor; 100 ng/mL; Invitrogen, Carlsbad, CA, USA) or PD98059 (MAPK suppressor; 15 μM; Beyotime). BDNF (0, 10, 20, 50, 100 and 200 ng/mL; PeproTech, London, UK)-treated GCs were used, for different times (12, 24, or 48 h). K252α (100 ng/mL) were used for 24 h, PD98059 (15 μM), for 30 min.

Zheng X., Chen L., Chen T., Cao M., Zhang B., Yuan C., Zhao Z., Li C, & Zhou X. (2023). The Mechanisms of BDNF Promoting the Proliferation of Porcine Follicular Granulosa Cells: Role of miR-127 and Involvement of the MAPK-ERK1/2 Pathway. Animals : an Open Access Journal from MDPI, 13(6), 1115.

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Transwell Bicameral Assay for BDNF Effect

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A Transwell bicameral chamber (Corning Costar) was used to co-culture the transfected 293T and ARPE-19 cells. The ARPE-19 cells were exposed to released BDNF continuously. Untransfected 293T cells or 293T cells transfected with pEGFP-N1-BDNF or pEGFP-N1 were seeded in the upper chamber of a Transwell plate 12 hours before transfection. ARPE-19 cells were seeded in the lower chamber of an adjacent well 7 hours before transfection of the 293T cells. The upper chamber was transferred to the corresponding adjacent ARPE-19 well at hour 0 after transfection. Purified BDNF (50 ng/mL or the same concentration as released BDNF; Thermo Fisher Scientific) was added to other ARPE-19 wells as a control. ARPE-19 cells were incubated for 24, 48, 72 or 96 hours before harvesting for 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and western blot assay (Figure 2).

Yan B.J., Wu Z.Z., Chong W.H, & Li G.L. (2016). Construction of a plasmid for human brain-derived neurotrophic factor and its effect on retinal pigment epithelial cell viability. Neural Regeneration Research, 11(12), 1981-1989.

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Differentiation of hiPSCs into Dopaminergic and Motor Neurons

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For neural induction, a modified method previously described was used (Chambers et al., 2009 (link)). hiPSCs were cultured in neural induction medium combining dual ALK inhibition (SB + LDN:10 μm SB431542 (Tocris Bioscience, Bristol, United Kingdom) and 0.2 μm LDN193189 (Stemgent, United Kingdom) in KSR media. For differentiating neuronal cells into neurons, increasing amounts of N2 (Gibco, Waltham, MA, United States) (25, 50, and 75%) was added to the KSR media maintaining LDN193189. On day 5–9 sonic hedgehog (SHH; Curis, Lexington, MA United States) was added in N2 media followed by the addition on day 9 of BDNF (PeproTech, London, United Kingdom), ascorbic acid (Sigma-Aldrich, St. Louis, MO, United States) and FGF8 (PeproTech, London, United Kingdom) and maturated on days 12–30 with BDNF, ascorbic acid, GDNF (PeproTech, London, United Kingdom), TGFβ3 (Sigma-Aldrich, St. Louis, MO, United States), cAMP (Sigma-Aldrich, St. Louis, MO, United States) for doparminergic subtype and retinoic acid (Sigma-Aldrich, St. Louis, MO, United States) for motor neuron cells.

Spitalieri P., Talarico R.V., Murdocca M., Fontana L., Marcaurelio M., Campione E., Massa R., Meola G., Serafino A., Novelli G., Sangiuolo F, & Botta A. (2018). Generation and Neuronal Differentiation of hiPSCs From Patients With Myotonic Dystrophy Type 2. Frontiers in Physiology, 9, 967.

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Imaging of Axonal Dynamics in Neurons

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Imaging was performed 7 d after adding PFFs at 37°C, 5% CO₂, with buffer containing 136 mM NaCl, 2.5 mM KCl, 2 mM CaCl₂, 1.3 mM MgCl₂,10 mM glucose, and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. For BDNF treatments, neurons were treated with 50 ng/ml BDNF (PeproTech, Rocky Hill, NJ) for 1 h before imaging. BDNF was also included in the imaging buffer. Epifluorescence imaging was performed with a Nikon TE-2000-E (Nikon, Tokyo, Japan) inverted microscope with a 60× oil immersion objective. Regions of thin axons at least 100 μm away from growth cones or adjacent cell bodies were chosen. Unless otherwise stated in the figure legends, images were captured every second for a total of 3 min at 300-ms exposure time. Spinning disk confocal images were captured with an Olympus IX81 (Center Valley, PA) inverted microscope and an iXon3 electron-multiplying charge-coupled device camera (Andor, South Windsor, CT) at the University of Pennsylvania Cell and Developmental Biology Microscopy Core Facility. Scanning confocal microscopy was performed using a TCS SP5 Visible-Upright Confocal Microscope (Leica Microsystems, Buffalo Grove, IL) at the University of Alabama-Birmingham Center for Neurodegeneration and Experimental Therapeutics.

Volpicelli-Daley L.A., Gamble K.L., Schultheiss C.E., Riddle D.M., West A.B, & Lee V.M. (2014). Formation of α-synuclein Lewy neurite–like aggregates in axons impedes the transport of distinct endosomes. Molecular Biology of the Cell, 25(25), 4010-4023.

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Differentiation of iPSC-Derived Neurons

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For differentiation of iPSC-derived neurons, NPCs were seeded at densities of 1.5 × 10⁵ cells onto matrigel coated 12-well plates in NPC medium. The next day medium was replaced by N2B27 medium containing 1 µm SAG, 100 µM AA, 2 ng/mL BDNF (Peprotech) and 2 ng/mL GDNF (Peprotech). At day 6 medium was exchanged to N2B27 medium containing 100 µM AA, 2 ng/mL BDNF, 2 ng/mL GDNF, 1 ng/mL TGFβ (Peprotech) and 100 µM dbcAMP. From day 6 to 10 5 ng/mL Activin A were added to the medium. Cells were replated onto matrigel coated coverslips at day 10 of differentiation.

Starost L., Lindner M., Herold M., Xu Y.K., Drexler H.C., Heß K., Ehrlich M., Ottoboni L., Ruffini F., Stehling M., Röpke A., Thomas C., Schöler H.R., Antel J., Winkler J., Martino G., Klotz L, & Kuhlmann T. (2020). Extrinsic immune cell-derived, but not intrinsic oligodendroglial factors contribute to oligodendroglial differentiation block in multiple sclerosis. Acta Neuropathologica, 140(5), 715-736.

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Differentiation of iPSCs into Mature Neurons

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We coaxed iPSCs from both control and SCN1A^M145T to differentiate into neural stem cells (NSCs) using Gibco^® PSC Neural Induction Medium (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s instructions. To obtain neurons, NSCs were then plated at a density of 5 × 10⁴ cells/cm² on dishes coated with Poly-D-Lysine (molecular weight 30,000–70,000) plus Laminin (both from Merck, Darmstadt, Germany) and cultured in Neuronal Differentiation Medium (NDMC), composed of Neurobasal Medium, 1× B27 supplement, 1× Glutamax, 1× CultureOne™ Supplement, 200 μM ascorbic acid and 0.2% Penicillin/Streptomycin (all from Thermo Fisher Scientific). NDMC was supplemented with GDNF at 10 ng/mL and BDNF at 20 ng/mL (both from PeproTech, London, UK) at NSCs plating; the concentration of GDNF and BDNF was lowered to 5 ng/m and 10 ng/m, respectively, at the first medium change. Subsequently, NDMC medium was supplemented with BDNF only, used at 5 ng/mL during the second medium change and at 2.5 ng/mL during the whole culture period. Neurons were kept in culture 28–35 days until they reached full maturation for subsequent analysis.

Scalise S., Zannino C., Lucchino V., Lo Conte M., Scaramuzzino L., Cifelli P., D’Andrea T., Martinello K., Fucile S., Palma E., Gambardella A., Ruffolo G., Cuda G, & Parrotta E.I. (2022). Human iPSC Modeling of Genetic Febrile Seizure Reveals Aberrant Molecular and Physiological Features Underlying an Impaired Neuronal Activity. Biomedicines, 10(5), 1075.

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Induction and Culture of Neural Precursors

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The early neural precursors used in our study were derived from the IMR90 cell-line generated by the transduction of female human fetal lung fibroblasts (ATCC, Manassas, VA, USA) with a cocktail of lentivirus-mediated human cDNAs (OCT4, SOX2, NANOG and LIN28). NP-iPS were induced in the growth media containing 500 ng/mL of Noggin (R&D Systems, Minneapolis, MN, USA); 10 nM transforming growth factor-β pathway inhibitor SB 431542 (Sigma-Aldrich, St. Louis, MO, USA); 10 μg/mL b FGF and 20 μg/mL BDNF (both from Pepro Tech, London, UK) [17 (link),25 (link),61 (link)]. A detailed description of the protocol, composition of growth media and solutions used for culturing the neural precursors were reported earlier by our group [25 (link)]. Briefly, NP-iPS were cultured in a medium composed of DMEM/F12 and Neurobasal media mixture (1:1) supplemented by B27 and N2 supplements (GIBCO, Life Technologies, Grand Island, NY, USA), l-glutamine (2 mM, Sigma-Aldrich), penicillin and streptomycin (50 U/mL, GIBCO), FGF (10 ng/mL), EGF (10 ng/mL), and BDNF (20 ng/mL) (PeproTech, London, UK). The media were replaced every second day. Prior to transplantation, NP-iPS were cultured for 7 days in the same medium, except for the omission of FGF and EGF factors.

Forostyak S., Forostyak O., Kwok J.C., Romanyuk N., Rehorova M., Kriska J., Dayanithi G., Raha-Chowdhury R., Jendelova P., Anderova M., Fawcett J.W, & Sykova E. (2020). Transplantation of Neural Precursors Derived from Induced Pluripotent Cells Preserve Perineuronal Nets and Stimulate Neural Plasticity in ALS Rats. International Journal of Molecular Sciences, 21(24), 9593.

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BDNF Infusion Modulates ACC Function

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For the BDNF treatment experiments, mice were randomly separated into two groups: one that was injected with 114 μl BDNF (0.0013 μg/μl, PeproTech, United States) into ACC for 3 days in succession and one that was injected with isovolumetric saline. For the BDNF infusion to the ACC, a unilateral stainless steel guide cannula (ID 0.14 mm, OD 0.3 mm, BIC-3, RWD Life Science, China) was implanted into the right ACC according to the following coordinates (anteroposterior: +0.73; dorsoventral: −1.75; mediolateral: −0.28) and secured to the skull with dental cement. Then, a mini osmotic pump (1003D, RWD Life Science, China) was embedded into subcutaneous tissue connected to the cannula, releasing the drug to the ACC at a 1.0 μl/hr for 3 days. After treatment, we performed behavioral assessments, electrophysiological tests and Western blot analyses.

Qi C., Chen A., Mao H., Hu E., Ge J., Ma G., Ren K., Xue Q., Wang W, & Wu S. (2022). Excitatory and Inhibitory Synaptic Imbalance Caused by Brain-Derived Neurotrophic Factor Deficits During Development in a Valproic Acid Mouse Model of Autism. Frontiers in Molecular Neuroscience, 15, 860275.

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