Journal: Cellular and Molecular Neurobiology

Article Title: Neurotrophic Activity of Cultured Cell Line U87 is Up-Regulated by Proline-Rich Polypeptide Complex and Its Constituent Nonapeptide

doi: 10.1007/s10571-015-0192-8

Figure Lengend Snippet: Western blotting analysis of NGF forms secreted by astrocytoma cell line U87. Supernatants from control and compound-treated cells were separated in SDS-PAGE, then transferred to nitrocellulose membrane. NGF forms were detected with the use of specific polyclonal antibodies anti-βNGF. Immunocomplexes were visualized using a NBT/BCIP substrate and analyzed densitometrically in Molecular Imager ChemiDoc MP Imaging System with Image Lab Softwere (BioRad). The representative immunoblot is presented

Article Snippet: Rabbit anti-BDNF polyclonal antibody was from Bioss (MA, USA).

Techniques: Western Blot, SDS Page, Imaging

Journal: Cell reports

Article Title: Variation of Human Neural Stem Cells Generating Organizer States In Vitro before Committing to Cortical Excitatory or Inhibitory Neuronal Fates

doi: 10.1016/j.celrep.2020.107599

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The following primary antibodies were used at the concentration indicated by manufacturer’s protocol: CaM Kinase II alpha (6G9) (NB100–1983), LMX1A (NBP1–81303) Novusbio; SYNAPSIN (106 001), HOMER (160 003) Synaptic System; EGFR (Ab231), FGFR1 phosphoY654 (Ab59194), TBR1 (Ab31940), REELIN (Ab18570), CYCLIN D1 (Ab10540), FGFR2 (Ab10648), BMPR1A (Ab38560) Abcam; HES1 (11988), p-SMAD1/5 (9516), CYCLIN D1 (2926), pERK1/2 (4370), FGFR1 (9740) Cell Signaling Technology; PAX6 (PRB-278P) BioLegend; NESTIN (MAB1259), OTX2 (AF1979), PDGFR alpha (AF1062; AF307), SOX2 (AF2018; MAB2018), SOX21 (AF3538), TuJ1 (MAB1195), EGFR (AF1280), O4 (MAB 1326) R&D Systems; GFAP (Z 0334) DAKO; HES5 (sc-13859), CUX1 (sc-13024), TLE4 (sc-9125), FGFR3 (sc-9007), LHX2 (sc-19344) Santa Cruz Biotechnology; FOXP2 (ABE73), TBR1 (AB2261), REELIN (MAB5366) Millipore; FOXG1 (M227) Takara/Clontech; anti GAD65/67 was kindly gifted by Dr. Christian Geis, Hans Berger Department of Neurology, Jena University Hospital, Germany ( ).

Techniques: Virus, Plasmid Preparation, Recombinant, Transfection, Antibody Labeling, In Vitro, Microarray, Gene Expression, Derivative Assay, Software

Journal: International Journal of Molecular Sciences

Article Title: Fingolimod Modulates Dendritic Architecture in a BDNF-Dependent Manner

doi: 10.3390/ijms21093079

Figure Lengend Snippet: Fingolimod-phosphate (FTY720-P) regulates neuronal architecture in a BDNF-dependent manner. ( A ) Representative Neurolucida tracings, used to perform the Sholl analysis of dendritic complexity for feGFP expressing neurons treated for 24h with: DMSO (black), 2nM FTY720-P (gray), DMSO + TrkBFc (dark blue) and 2nM FTY720-P + TrkBFc (light blue). Scale bar: 100 μm. ( B ) Sholl analysis displayed as number of dendritic intersections against distance from the cell body for DMSO (black), 2nM FTY720-P (gray), DMSO + TrkBFc (dark blue) and 2nM FTY720-P + TrkBFc (light blue) and ( C ) total dendritic complexity of all treatment groups. F value in ( B ) refers to comparison between all the 4 treatment groups. ( D ) Dendritic spine densities for DMSO (black), 2nM FTY720-P (gray), DMSO + TrkBFc (dark blue) and 2nM FTY720-P + TrkBFc (light blue) calculated using segments of secondary dendritic branches as shown in ( E ) feGFP panel. The Syn panel displays the corresponding staining of the pre-synaptic marker SynapsinI/II and the merge panel shows the images with overlapping SynapsinI/II puncta (red, pre-synapse) to its matching feGFP dendrite segment (green, post-synapse). The arrows point to coinciding puncta, indicative of mature synapse between the post and pre-synaptic compartments. Scale bar: 5μm. ( F ) The graph compares the fraction of SynapsinI/II positive feGFP labelled spines for DMSO (black), 2nM FTY720-P (gray), DMSO + TrkBFc (dark blue) and 2nM FTY720-P + TrkBFc (light blue). ( G ) Representative fields of view (FOV) of the DMSO, 2nM FTY720-P, DMSO + TrkBFc and 2nM FTY720-P + TrkBFc treated hippocampal cultures stained for c-fo s . Scale bar: 100μm. ( H ) Quantification of the proportion of c-Fos expressing neurons represented as normalized fraction for DMSO (black), 2nM FTY720-P (gray), DMSO + TrkBFc (dark blue) and 2nM FTY720-P + TrkBFc (light blue) treated cultures. All graphs represent data as mean + SEM. Numbers in the bars show either total number of neurons or of FOV analyzed, obtained from ≥3 sets of independent experiments. Two-way ANOVA followed by Bonferroni post-hoc test was used in ( B ). For ( C , D , F ) and ( H ) one-way ANOVA with Bonferroni post-hoc was used. Denotations for significance are * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: Recombinant Human BDNF protein (R&D systems, Minneapolis, MN, USA) was dissolved in sterile PBS with 0.1% BSA at a concentration of 50 ng/μL.

Techniques: Expressing, Comparison, Staining, Marker

Journal: International Journal of Molecular Sciences

Article Title: Fingolimod Modulates Dendritic Architecture in a BDNF-Dependent Manner

doi: 10.3390/ijms21093079

Figure Lengend Snippet: Treatment with the non-phosphorylated Fingolimod (FTY720) modulates neuronal architecture. ( A ) | Representative Neurolucida tracings from feGFP positive hippocampal neurons used for the Sholl analysis from cultures treated either with DMSO or 10nM FTY20 for 24 h. Scale bar: 100μm. ( B ) | Dendritic complexity shown by the number of dendritic intersections plotted against the distance from the soma for DMSO (black) and FTY720 (blue) treated neurons. The F value shows the statistical comparison between the two groups. The inset graph represents total dendritic complexity upon treatment with DMSO (black) and FTY720 (blue). ( C ) | Total dendritic length for DMSO (black) and FTY720 (blue) treated neurons. ( D ) | Representative stretches from dendrites of eGFP transfected hippocampal neurons showing dendritic spine protrusions, treated either with DMSO or FTY720 for 24h. Scale bar: 5μm. ( E ) | The graph shows dendritic spine density for DMSO (black) and FTY720 (blue) treated neurons . ( F ) | Representative images of fields of view (FOV) from primary hippocampal cultures stained with anti phospho-ERK1/2 antibody, 30 min post-application of one of the following: DMSO, 10nM FTY720, DMSO_100, 100nM FTY720, DMSO_100 + TrkB-Fc, 100nM FTY720 + TrkB-Fc or 40ng recombinant BDNF protein as a positive control. Scale bar: 100μm. ( G ) | The graph displays the fraction of pERK1/2 expressing neurons relative to the total number of MAP2 + neurons. The data is normalized to the respective controls and compared between the different treatment groups: DMSO (black), 10nM FTY720 (light blue solid), DMSO100 (gray), 100nM FTY720 (dark blue solid), 40ng recombinant BDNF (magenta), DMSO100 + TrkB-Fc (gray open), 100nM FTY720 + TrkB-Fc (dark blue open). All data is plotted as mean + SEM. Numbers in the bars show total number of neurons or FOV analyzed, obtained from ≥3 sets of independent experiments. Two-way ANOVA followed by Bonferroni post-hoc test was used in ( B ). For ( B ) total intersections, ( C ) and ( E ) unpaired Student’s t-test and for ( G ) one-way ANOVA with Bonferroni post-hoc was used. Denotations for significance are * p < 0.05, *** p < 0.001, **** p < 0.0001.

Article Snippet: Recombinant Human BDNF protein (R&D systems, Minneapolis, MN, USA) was dissolved in sterile PBS with 0.1% BSA at a concentration of 50 ng/μL.

Techniques: Comparison, Transfection, Staining, Recombinant, Positive Control, Expressing

Journal: The Journal of Neuroscience

Article Title: Local and Target-Derived Brain-Derived Neurotrophic Factor Exert Opposing Effects on the Dendritic Arborization of Retinal Ganglion CellsIn Vivo

doi: 10.1523/jneurosci.22-17-07639.2002

Figure Lengend Snippet: Figure 1. Altering endogenous retinal and tectal BDNF levels in vivo. Diagrams representing a transverse view of a Xenopus tadpole brain and eye illustrate experimental procedures (see Materials and Methods). RGCs are depicted in red, relative endogenous BDNF expression levels (Cohen-Cory et al., 1996) are depicted in blue, and exogenously applied factors are depicted in green. A, Effects of altered tectal neurotrophins on RGC dendritic arborization. Control, anti-BDNF, or BDNF-treated green fluorescent microspheres were injected into the stage 38 tadpole tectum. At stage 42, RGCs were retrogradely labeled by injecting rhodamine–dextran in the contralateral tectum. At stage 45, dendritic morphologies of double-labeled RGCs were evaluated. A low-power view of a tadpole eye shows green fluorescent microspheres retrogradely transported to the retinal ganglion cell layer, where a rhodamine–dextran-labeled RGC soma can also be visualized (lines denote lens and eye periphery). Scale bar, 50 m. A single-plane, high-power view of a stage 45 retina reveals a rhodamine–dextran-labeled RGC with internalized green fluorescent microspheres. Scale bar, 5 m. B, Effects of altered retinal neurotrophins on RGC dendritic arborization. Control, anti-BDNF, or BDNF-treated microspheres were injected into the stage 38 tadpole retina, and then RGCs were retrogradely labeled at stage 42. The low-power view shows rhodamine–dextran-labeled RGCs and green fluorescent microspheres restricted within the tadpole eye. Scale bar, 200 m. The single-plane, high-power view of a stage 45 retina reveals the morphology of a rhodamine– dextran-labeled RGC surrounded by green fluorescent microspheres. Scale bar, 5 m. C, Effects of altered retinal neurotrophins on RGC axonal arborization in the tectum. Control, anti-BDNF, or BDNF-treated microspheres were injected into the stage 43 tadpole retina, and the morphology of DiI- or YFP-labeled RGC axon arbors was visualized 24 and 48 hr later. Confocal microscope images of a control RGC axon at 0 and 24 hr demonstrate normal RGC axon arborization dynamics. Scale bar, 20 m.

Article Snippet: Recombinant human BDNF (rhBDNF) was kindly provided by Amgen (Thousand Oaks, CA), recombinant human neurotrophin-4 (NT-4) was generously provided by Genentech (South San Francisco, CA), and anti-rhBDNF neutralizing antibody (mouse IgG1) was obtained from R & D Systems (Minneapolis, MN).

Techniques: In Vivo, Expressing, Control, Injection, Labeling, Microscopy

Journal: The Journal of Neuroscience

Article Title: Local and Target-Derived Brain-Derived Neurotrophic Factor Exert Opposing Effects on the Dendritic Arborization of Retinal Ganglion CellsIn Vivo

doi: 10.1523/jneurosci.22-17-07639.2002

Figure Lengend Snippet: Figure 2. Tectal BDNF retrogradely enhances RGC dendritic arborization. To determine whether tectal BDNF influences RGC dendritic arborization within the retina, tadpoles received tectal injections of microspheres treated with control, BDNF, or anti-BDNF function-blocking antibodies. Microsphere-containing neurons colabeled with rhodamine–dextran were analyzed morphologically (Fig. 1A). A, Image reconstructions of two rhodamine-labeled RGCs with simple and complex dendritic arbors illustrate differences in dendritic arbor morphologies. B, Images of RGC dendritic arbors reveal that increasing tectal BDNF enhances RGC dendritic arborization, whereas neutralizing endogenous tectal BDNF with function-blocking antibodies reduces RGC dendritic arborization. C, Quantitative analysis reveals that primary dendrite number, branch tip number, branch tips per primary dendrite, and overall dendritic length were significantly enhanced by increasing tectal BDNF and reduced by injecting anti-BDNF into the optic tectum. Scale bar, 5 m. Error bars indicate SEM. p 0.05; p 0.01; p 0.001.

Article Snippet: Recombinant human BDNF (rhBDNF) was kindly provided by Amgen (Thousand Oaks, CA), recombinant human neurotrophin-4 (NT-4) was generously provided by Genentech (South San Francisco, CA), and anti-rhBDNF neutralizing antibody (mouse IgG1) was obtained from R & D Systems (Minneapolis, MN).

Techniques: Control, Blocking Assay, Labeling

Journal: The Journal of Neuroscience

Article Title: Local and Target-Derived Brain-Derived Neurotrophic Factor Exert Opposing Effects on the Dendritic Arborization of Retinal Ganglion CellsIn Vivo

doi: 10.1523/jneurosci.22-17-07639.2002

Figure Lengend Snippet: Figure 4. Retinal BDNF inhibits RGC den- dritic arborization in a dose-dependent man- ner. To determine whether RGCs are sensi- tive to the concentration of BDNF in the retina, Xenopus retinas were microinjected with 1–100 ng/l BDNF or control micro- spheres at the onset of dendritic arborization. Quantitative measures of dendritic arbor morphology revealed a dose-dependent re- sponse to BDNF. The highest concentration of BDNF most dramatically decreased pri- mary dendrite number, branch tip number, tips per dendrite, and dendrite length versus control. Error bars indicate SEM. p 0.05; p 0.01; p 0.001.

Article Snippet: Recombinant human BDNF (rhBDNF) was kindly provided by Amgen (Thousand Oaks, CA), recombinant human neurotrophin-4 (NT-4) was generously provided by Genentech (South San Francisco, CA), and anti-rhBDNF neutralizing antibody (mouse IgG1) was obtained from R & D Systems (Minneapolis, MN).

Techniques: Concentration Assay, Control

Journal: The Journal of Neuroscience

Article Title: Local and Target-Derived Brain-Derived Neurotrophic Factor Exert Opposing Effects on the Dendritic Arborization of Retinal Ganglion CellsIn Vivo

doi: 10.1523/jneurosci.22-17-07639.2002

Figure Lengend Snippet: Figure 5. RGC dendritic arborization is temporally sensitive to increased reti- nal BDNF levels. To determine whether RGCs were sensitive to enhanced retinal BDNF in a stage-dependent manner, control or BDNF-treated microspheres were injected into Xenopus retinas at stage 38 or 42. A, The morphology of RGC dendritic arbors revealed a stage- dependent response to increased retinal BDNF levels. B–C, Quantitative analy- sis of dendritic differentiation indicates that earlier exposure to exogenous BDNF (stages 38–45) inhibited den- dritic arborization more dramatically than later exposure to BDNF (stages 42–45). Primary dendrite number as well as dendritic branching was signifi- cantly decreased by altering retinal BDNF starting at stage 38 (B), whereas altering retinal BDNF levels from stage 42 onward (C) selectively reduced den- dritic branching without affecting pri- mary dendrite number. Error bars indi- cate SEM. p 0.05; p 0.001. Scale bar, 10 m.

Article Snippet: Recombinant human BDNF (rhBDNF) was kindly provided by Amgen (Thousand Oaks, CA), recombinant human neurotrophin-4 (NT-4) was generously provided by Genentech (South San Francisco, CA), and anti-rhBDNF neutralizing antibody (mouse IgG1) was obtained from R & D Systems (Minneapolis, MN).

Techniques: Control, Injection

Journal: The Journal of Neuroscience

Article Title: Local and Target-Derived Brain-Derived Neurotrophic Factor Exert Opposing Effects on the Dendritic Arborization of Retinal Ganglion CellsIn Vivo

doi: 10.1523/jneurosci.22-17-07639.2002

Figure Lengend Snippet: Figure 6. RGC axon arbor complexity is unaffected by retinal BDNF levels. To deter- mine whether retinal BDNF influences RGC axon arborization at a distance, tadpoles were intraocularly injected with control, BDNF-, or anti-BDNF-treated microspheres, and the re- sulting changes in RGC axon arbor dynamics were compared with tectally applied BDNF (Cohen-Cory and Fraser, 1995; Lom and Co- hen-Cory, 1999). A, Individual RGC axon ar- bor morphologies of control, retinal BDNF, and tectal BDNF at 0 and 24 hr after treat- ment demonstrate that only tectally applied BDNF significantly alters RGC axon ar- borization. B, C, Altering retinal BDNF lev- els had no significant effects on RGC axon arbor complexity as measured by the in- crease in total branch number (B) and total arbor length (C) 24 and 48 hr after treatment ( p 0.05). Error bars indicate SEM. Scale bar, 20 m.

Article Snippet: Recombinant human BDNF (rhBDNF) was kindly provided by Amgen (Thousand Oaks, CA), recombinant human neurotrophin-4 (NT-4) was generously provided by Genentech (South San Francisco, CA), and anti-rhBDNF neutralizing antibody (mouse IgG1) was obtained from R & D Systems (Minneapolis, MN).

Techniques: Injection, Control

Journal:

Article Title: Up-Regulation of Insulin-Like Growth Factor-II Expression Is a Feature of TrkA but Not TrkB Activation in SH-SY5Y Neuroblastoma Cells

doi:

Figure Lengend Snippet: Induction of early response genes. Cells were cultured for 3 days in the presence (1 μg/ml) and absence of tetracycline in RPMI containing 10% FBS and were stimulated with cognate ligands at a concentration of 100 ng/ml. Profiles of the early response genes induced by NGF and BDNF are similar. The response was more sustained in the absence of tetracycline. Fifteen micrograms of total RNA was electrophoresed in 1.2% formaldehyde gel and subsequently transferred to nitrocellulose membrane. tet +: 1 μg tetracycline/ml; tet −: 0 μg tetracycline/ml.

Article Snippet: They were then harvested after stimulation with 100 ng/ml of 2.5S mouse NGF (Upstate Biotechnology, Lake Placid, NY) or recombinant human BDNF (Promega, Madison, WI) for 5 minutes.

Techniques: Cell Culture, Concentration Assay

Journal:

Article Title: Up-Regulation of Insulin-Like Growth Factor-II Expression Is a Feature of TrkA but Not TrkB Activation in SH-SY5Y Neuroblastoma Cells

doi:

Figure Lengend Snippet: Changes in lineage-dependent differentiation markers. Cells were cultured in RPMI containing 10% FBS and treated with 100 ng/ml of NTs for 5 days in the presence (1 μg/ml) or absence of tetracycline. A: Immunoblot analysis of neural cell adhesion molecule (NCAM) was performed with mouse monoclonal antibody to NCAM. Forty micrograms of proteins was electrophoresed in 8% SDS-PAGE. TrkA-transfected clones showed decreases in both 185-kd and 145-kd isoforms of NCAM with NGF treatment. Reduced NCAM signal in TB3 with high trkB is due to an underloading of the protein. Immunoblot analysis of GAP-43 showed increased expression in TA25, TA88, TB3, and TB8 cells after NT treatment for 5 days in the absence of tetracycline. The GAP-43 expression level was not altered in E2 cells by NGF or BDNF. B: Northern blot analysis of bcl-2, IGF-II, and chromogranin A (CGA) expression. NGF and BDNF commonly increased bcl-2 expression of trkA and trkB transfected clones. Robust increases in IGF-II expression marked a characteristic feature of trkA transfected clones. Chromogranin A expression increased in one (TB3) of the trkB transfected clones. C: The results of radioimmunoassay using two-site immunoradiometric assay of IGF-II, which shows three- to sixfold increases in the concentration of IGF-II in the cell culture media in which TA25 and TA88 cells were grown in the absence of tetracycline, respectively. The concentration was adjusted according to the absolute cell numbers. The data were represented as a percentage of control (without NT treatment). D: Analysis of norepinephrine in media after 5 days of NT treatment in experimental and control cells by high-pressure liquid chromatography. An approximately twofold increase in norepinephrine concentration was found in TA25 and TA88 when the cells were treated with NGF (100 ng/ml) for 5 days in the absence of tetracycline. tet −: 0 μg tetracycline/ml.

Article Snippet: They were then harvested after stimulation with 100 ng/ml of 2.5S mouse NGF (Upstate Biotechnology, Lake Placid, NY) or recombinant human BDNF (Promega, Madison, WI) for 5 minutes.

Techniques: Cell Culture, Western Blot, SDS Page, Transfection, Clone Assay, Expressing, Northern Blot, RIA Assay, Immunoradiometric Assay, Concentration Assay, High Performance Liquid Chromatography

Journal:

Article Title: Up-Regulation of Insulin-Like Growth Factor-II Expression Is a Feature of TrkA but Not TrkB Activation in SH-SY5Y Neuroblastoma Cells

doi:

Figure Lengend Snippet: Effects of NGF and BDNF on protection from nutrient deprivation. Cells were cultured for 5 days in RPMI containing 0.5% serum. Viable and nonviable cells were counted based on trypan blue exclusion. A: Nutrient deprivation was associated with more exaggerated differentiation after NGF and BDNF treatment. B: Fraction of viable cells represented as a percentage of the control (without NT treatment). Low trkA protected the cells to a level comparable to that of high trkA in each clone with NGF treatment, whereas trkB transfected clones showed a dose-dependent effect. tet +: 1 μg tetracycline/ml; tet −: 0 μg tetracycline/ml.

Article Snippet: They were then harvested after stimulation with 100 ng/ml of 2.5S mouse NGF (Upstate Biotechnology, Lake Placid, NY) or recombinant human BDNF (Promega, Madison, WI) for 5 minutes.

Techniques: Cell Culture, Transfection, Clone Assay