Journal: Pharmaceuticals

Article Title: A Safe GDNF and GDNF/BDNF Controlled Delivery System Improves Migration in Human Retinal Pigment Epithelial Cells and Survival in Retinal Ganglion Cells: Potential Usefulness in Degenerative Retinal Pathologies

doi: 10.3390/ph14010050

Figure Lengend Snippet: Microsphere (MS) characterization. Morphological evaluation by scanning electron microscopy (SEM) and particle size distribution. Blank MSs (MSs); MSs/VitaminE(20) (MSs-E20); MSs/VitaminE(40) (MSs-E40) GDNF/VitE(20)-loaded PLGA MSs (MSs-GE20); GDNF/BDNF/VitE(40)-loaded PLGA microspheres (MSs-GBE40). SEM investigation showed the presence of spherical particles with comparable and regular size distributions, which were confirmed by particle size measurements. White arrows: pores on the MS surfaces. Scale bar: 10 µm.

Article Snippet: The first formulation was performed suspending 20 μg of recombinant human GDNF (R&D Systems, Minneapolis, MN, USA) in 20 μL of VitE (Sigma-Aldrich, Schnelldorf, Germany).

Techniques: Electron Microscopy

Journal: Pharmaceuticals

Article Title: A Safe GDNF and GDNF/BDNF Controlled Delivery System Improves Migration in Human Retinal Pigment Epithelial Cells and Survival in Retinal Ganglion Cells: Potential Usefulness in Degenerative Retinal Pathologies

doi: 10.3390/ph14010050

Figure Lengend Snippet: Release profiles of NTFs from PLGA/VitE matrices are presented expressed as cumulative ng NTF/mg MSs, cumulative % of total loaded protein and the release rate of NTF (pg NTF/mg MSs/day). ( A ) GDNF release from GDNF-loaded MSs. ( B ) GDNF (■) and BDNF (○) release from GDNF/BDNF-loaded MSs. Release media: PBS (pH 7.4) with 1.0% of BSA and 0.02% sodium azide.

Article Snippet: The first formulation was performed suspending 20 μg of recombinant human GDNF (R&D Systems, Minneapolis, MN, USA) in 20 μL of VitE (Sigma-Aldrich, Schnelldorf, Germany).

Techniques:

Journal: Pharmaceuticals

Article Title: A Safe GDNF and GDNF/BDNF Controlled Delivery System Improves Migration in Human Retinal Pigment Epithelial Cells and Survival in Retinal Ganglion Cells: Potential Usefulness in Degenerative Retinal Pathologies

doi: 10.3390/ph14010050

Figure Lengend Snippet: Safety and apoptosis of the delivery of neurotrophins by microspheres. MSs-GE and MSs-GBE showed no alterations in ARPE-19 ( A ) and RF/6A ( B ) cell viability measured by MTT. Blank PLGA MSs and PLGA/Vit E MSs (MSs-E20 and MSs-E40) controls also showed similar cell viability values in ARPE-19 ( A ) and RF/6A cells ( B ). Apoptosis was detected by Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling TUNEL (red) and nuclei were stained with DAPI (blue). TUNEL positive cells were only found in ARPE-19 ( E ) and RF/6A ( J ) cells after NaIO 3 treatment for 24 h (1500 µg/mL for ARPE-19 cells and 500 µg/mL for RF/6A). TUNEL signal was absent in ARPE-19 ( C , D , F , G ) and RF/6A ( H , I , K , L ) cells after treatment with MSs_80, MSs-GE20_80 and MSs-GBE40_80. Blank MSs (MSs_80); GDNF/VitE(20)-loaded PLGA MSs (MSs-GE20_80); GDNF/BDNF/VitE(40)-loaded PLGA microspheres (MSs-GBE40_80). Scale bar 20 µm. n = 4 for MTT assay and n = 3 for TUNEL detection.

Article Snippet: The first formulation was performed suspending 20 μg of recombinant human GDNF (R&D Systems, Minneapolis, MN, USA) in 20 μL of VitE (Sigma-Aldrich, Schnelldorf, Germany).

Techniques: End Labeling, TUNEL Assay, Staining, MTT Assay

Journal: Pharmaceuticals

Article Title: A Safe GDNF and GDNF/BDNF Controlled Delivery System Improves Migration in Human Retinal Pigment Epithelial Cells and Survival in Retinal Ganglion Cells: Potential Usefulness in Degenerative Retinal Pathologies

doi: 10.3390/ph14010050

Figure Lengend Snippet: Wound closure area in ARPE-19 cells. MSs-GBE (−) treated cells showed a more closed wound area than MSs-GE (−) treated cells both at 24 h ( A ) and 30 h ( B ) from scratch ( p <0.05 and p < 0.01, respectively) and than MSs-E20_40 ( - - - ) at 30 h ( B , p < 0.05). Graphs ( C – F ) and representative images ( G , H ) show a different pattern in timeline migration between MSs-GBE and MSs-GE treated groups in ARPE-19 cells at 0, 7, 24, 30, 48 and 54 h after scratching. Black dotted lines indicate the wound borders at the different time points and treatments. Blank MSs (MSs_20) and (MSs_40); GDNF/VitE(20)-loaded PLGA MSs (MSs-GE20_40); GDNF/BDNF/VitE(40)-loaded PLGA microspheres (MSs-GBE40_20). Scale bar: 100 µm. n = 6–8. * p < 0.05 and ** p < 0.01 MSs-GBE vs. MSs-GE; † p < 0.05 MSs-GBE vs. MSs-E40_20.

Article Snippet: The first formulation was performed suspending 20 μg of recombinant human GDNF (R&D Systems, Minneapolis, MN, USA) in 20 μL of VitE (Sigma-Aldrich, Schnelldorf, Germany).

Techniques: Migration

Journal: Pharmaceuticals

Article Title: A Safe GDNF and GDNF/BDNF Controlled Delivery System Improves Migration in Human Retinal Pigment Epithelial Cells and Survival in Retinal Ganglion Cells: Potential Usefulness in Degenerative Retinal Pathologies

doi: 10.3390/ph14010050

Figure Lengend Snippet: Wound closure in RF/6A cells represented by scatter plot and representative images. No statistically significant differences were found at 24 and 30 h ( A , B ) post-scratching. Moreover, wound closure pattern were similar for both treatments, MSs-GE (−) and MSs-GBE (−) in RF/6A cells at 0, 7, 24, 30, 48 and 54 h after scratch as shown in graphs ( C – F ) and representative images ( G , H ). Black dotted lines indicate the wound borders at the different time points and treatments. Black dotted lines indicate the wound borders at the different time points and treatments. Blank MSs (MSs_20) and (MSs_40); GDNF/VitE(20)-loaded PLGA MSs (MSs-GE20_40); GDNF/BDNF/VitE(40)-loaded PLGA microspheres (MSs-GBE40_20). Scale bar: 100 µm. n = 7–9.

Article Snippet: The first formulation was performed suspending 20 μg of recombinant human GDNF (R&D Systems, Minneapolis, MN, USA) in 20 μL of VitE (Sigma-Aldrich, Schnelldorf, Germany).

Techniques:

Journal: Pharmaceuticals

Article Title: A Safe GDNF and GDNF/BDNF Controlled Delivery System Improves Migration in Human Retinal Pigment Epithelial Cells and Survival in Retinal Ganglion Cells: Potential Usefulness in Degenerative Retinal Pathologies

doi: 10.3390/ph14010050

Figure Lengend Snippet: Histology (hematoxylin and eosin staining) of retinas one week after intravitreal injection. ( A ) Whole eye section showing an optic nerve (ON) and peripheral (p) framed areas observed. Retinal section from eye injected with saline ( B , C ), sodium iodate ( D , E ), MSs ( F , G ), MSs-E40 ( H , I ), MSs-GBE40 ( J , K ). No alterations (swelling, vacuoles, missed cells) were observed in any studied group. Scale bar: 1 mm ( A ) and 100 µm ( B – I ). Blank MSs (MSs); MSs/VitaminE(40) (MSs-E40), GDNF/BDNF/VitE(40)-loaded PLGA microspheres (MSs-GBE40). Abbreviations: RPE: retinal pigment epithelium, OS: outer segments, ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer, ON: optic nerve, p: periphery.

Article Snippet: The first formulation was performed suspending 20 μg of recombinant human GDNF (R&D Systems, Minneapolis, MN, USA) in 20 μL of VitE (Sigma-Aldrich, Schnelldorf, Germany).

Techniques: Staining, Injection, Saline

Journal: Pharmaceuticals

Article Title: A Safe GDNF and GDNF/BDNF Controlled Delivery System Improves Migration in Human Retinal Pigment Epithelial Cells and Survival in Retinal Ganglion Cells: Potential Usefulness in Degenerative Retinal Pathologies

doi: 10.3390/ph14010050

Figure Lengend Snippet: Immunofluorescent staining with anti-NeuN in mice retinas. NeuN (red) labeling is observed in a few cells in INL and mainly in GCL. Images show no alterations in GCL after intravitreal injection of saline ( A ), MSs ( C ), MSs-E40 ( D ) and MSs-GEB40 ( E ). Alterations in GCL were only found in sodium iodate injected animals ( B ). Nuclei of retinal cells were stained with DAPI (blue). Blank MSs (MSs); MSs/VitaminE(40) (MSs-E40), GDNF/BDNF/VitE(40)-loaded PLGA microspheres (MSs-GBE40). Scale bar: 20 µm. Abbreviations: RPE: retinal pigment epithelium, ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer.

Article Snippet: The first formulation was performed suspending 20 μg of recombinant human GDNF (R&D Systems, Minneapolis, MN, USA) in 20 μL of VitE (Sigma-Aldrich, Schnelldorf, Germany).

Techniques: Staining, Labeling, Injection, Saline

Journal: Pharmaceuticals

Article Title: A Safe GDNF and GDNF/BDNF Controlled Delivery System Improves Migration in Human Retinal Pigment Epithelial Cells and Survival in Retinal Ganglion Cells: Potential Usefulness in Degenerative Retinal Pathologies

doi: 10.3390/ph14010050

Figure Lengend Snippet: TUNEL staining of retinal tissue. Representative micrographs of retina sections were evaluated for apoptosis by TUNEL assay at 1 week after intravitreal injection. ( A ) Whole eye section shows the retinal areas observed. ( B ) Retinal section from eye injected with saline without TUNEL positive cells. ( C ) Retinal section from eye injected with sodium iodate, a control positive of apoptosis. TUNEL-positive cells were identified with red fluorescence retinal section from eyes injected with MSs, MSs-E40 and MSs-GBE40 ( D , E , F , respectively). TUNEL-positive cells were not found in eyes injected with PLGA and MSs. Nuclei of retinal cells were stained with DAPI (blue). Blank MSs (MSs); MSs/VitaminE(40) (MSs-E40), GDNF/BDNF/VitE(40)-loaded PLGA microspheres (MSs-GBE40). Abbreviations: ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer. Scale bar: 20 µm.

Article Snippet: The first formulation was performed suspending 20 μg of recombinant human GDNF (R&D Systems, Minneapolis, MN, USA) in 20 μL of VitE (Sigma-Aldrich, Schnelldorf, Germany).

Techniques: TUNEL Assay, Staining, Injection, Saline, Control, Fluorescence

Journal: Pharmaceuticals

Article Title: A Safe GDNF and GDNF/BDNF Controlled Delivery System Improves Migration in Human Retinal Pigment Epithelial Cells and Survival in Retinal Ganglion Cells: Potential Usefulness in Degenerative Retinal Pathologies

doi: 10.3390/ph14010050

Figure Lengend Snippet: Bioactivity of GDNF/BDNF was demonstrated at 1 h, 4, and 7 weeks of the release study, increasing RGC survival by 70%, 43%, and 64%, respectively, compared to Blank MSs. Blank MSs (MSs); GDNF/BDNF/VitE(40)-loaded PLGA microspheres (MSs-GBE40).

Article Snippet: The first formulation was performed suspending 20 μg of recombinant human GDNF (R&D Systems, Minneapolis, MN, USA) in 20 μL of VitE (Sigma-Aldrich, Schnelldorf, Germany).

Techniques:

Journal: The Journal of Neuroscience

Article Title: Neuroprotection Mediated by Glial Cell Line-Derived Neurotrophic Factor: Involvement of a Reduction of NMDA-Induced Calcium Influx by the Mitogen-Activated Protein Kinase Pathway

doi: 10.1523/jneurosci.21-09-03024.2001

Figure Lengend Snippet: Figure 1. Comparison of protein expression of GDNF and GDNF receptor complex between murine cerebral cortex and cultured cortical neurons and astrocytes. Total RNAs of murine astrocytic (Ast) and neuronal (Neur) cortical cultures and murine cerebral cortex (Cx) were extracted and reverse transcribed using a poly-dT primer. A, D, G, PCR amplification was performed then using a specific set of primers for GDNF (A), GFRa-1 (D), and c-Ret (G). B, E, H, Western blot was performed on a lysate derived from murine cerebral cortex, neurons, or astrocyte cultures showing GDNF (B) and GDNF receptor expression (E, H). C, F, I, Immunocytochemistry analysis of GDNF and GDNF receptor components in cultured astrocytes and neurons. Bright-field photomicrographs of either pure cortical astrocytes or pure cortical neu- rons in culture, after fixation and peroxidase staining for antibodies raised against GDNF (C), GFRa-1 (F), and c-Ret (I).

Article Snippet: Recombinant human GDNF (rhGDNF) was obtained from R&D Systems (Oxon, UK), and antibodies raised against GDNF, GFRa-1, c-Ret, extracellular signal-regulated kinase 1 (ERK1), phospho-ERKs (p-ERKs), p-c-Jun N-terminal protein kinase (p-JNK), and p-38 were purchased from Santa Cruz (Heidelberg, Germany).

Techniques: Comparison, Expressing, Cell Culture, Reverse Transcription, Western Blot, Derivative Assay, Immunocytochemistry, Staining

Journal: The Journal of Neuroscience

Article Title: Neuroprotection Mediated by Glial Cell Line-Derived Neurotrophic Factor: Involvement of a Reduction of NMDA-Induced Calcium Influx by the Mitogen-Activated Protein Kinase Pathway

doi: 10.1523/jneurosci.21-09-03024.2001

Figure Lengend Snippet: Figure 3. rhGDNF does not protect neurons against apoptotic cell death. To induce apoptosis, near pure cortical neuronal cultures (DIV 7) were transferred to a serum-deficient medium (SD). A, B, 24 hr after serum deprivation, the neurons were fixed and stained with DAPI to observe DNA condensation (A) or the DNA of cortical neurons was extracted and loaded onto an agarose gel to evidence DNA fragmentation (B). C, D, Neuronal death percentage was assessed after 24 hr by trypan blue dye staining (mean 6 SEM; n 5 12) after SD (C) or by LDH release (mean 6 SEM; n 5 12) after exposure of mixed cortical cultures (14 DIV) to staurosporine 200 nM (Stp) (D). SD and Stp exposure were performed in the presence ( gray bars) or absence (white bars) of rhGDNF and with or without 1 mg/ml cycloheximide (CHX). In both apoptotic paradigms, MK-801 was systematically added to block the secondary activation of NMDA receptors. * indicates significantly different from SD or STP by ANOVA, followed by Bonferroni–Dunn’s test ( p , 0.05).

Article Snippet: Recombinant human GDNF (rhGDNF) was obtained from R&D Systems (Oxon, UK), and antibodies raised against GDNF, GFRa-1, c-Ret, extracellular signal-regulated kinase 1 (ERK1), phospho-ERKs (p-ERKs), p-c-Jun N-terminal protein kinase (p-JNK), and p-38 were purchased from Santa Cruz (Heidelberg, Germany).

Techniques: Staining, Agarose Gel Electrophoresis, Blocking Assay, Activation Assay

Journal: The Journal of Neuroscience

Article Title: Neuroprotection Mediated by Glial Cell Line-Derived Neurotrophic Factor: Involvement of a Reduction of NMDA-Induced Calcium Influx by the Mitogen-Activated Protein Kinase Pathway

doi: 10.1523/jneurosci.21-09-03024.2001

Figure Lengend Snippet: Figure 2. GDNF induces a phosphorylation of ERKs. Mouse primary cortical neurons (A) or astrocytes (B) were treated with rhGDNF (10 ng/ml) for the indicated times. Aliquots of whole-cell lysates were sepa- rated by SDS-PAGE, transferred to membranes, and incubated with the anti-p-ERKs antibody or anti-total ERK antibody. The immunoblots are representative of three independent experiments.

Article Snippet: Recombinant human GDNF (rhGDNF) was obtained from R&D Systems (Oxon, UK), and antibodies raised against GDNF, GFRa-1, c-Ret, extracellular signal-regulated kinase 1 (ERK1), phospho-ERKs (p-ERKs), p-c-Jun N-terminal protein kinase (p-JNK), and p-38 were purchased from Santa Cruz (Heidelberg, Germany).

Techniques: Phospho-proteomics, SDS Page, Incubation, Western Blot

Journal: The Journal of Neuroscience

Article Title: Neuroprotection Mediated by Glial Cell Line-Derived Neurotrophic Factor: Involvement of a Reduction of NMDA-Induced Calcium Influx by the Mitogen-Activated Protein Kinase Pathway

doi: 10.1523/jneurosci.21-09-03024.2001

Figure Lengend Snippet: Figure 4. rhGDNF is neuroprotective against NMDA-induced neuronal death. A, Cultures were exposed for 24 hr to AMPA (10 mM) or kainate (50 mM) (with 10 mM MK-801 added to block secondary NMDA receptor- mediated toxicity), without (white bars) or with ( gray bars) 10 ng/ml rhGDNF. Medium LDH was assessed at the end of the exposure to excitotoxins (mean 6 SEM; n 5 10 cultures per condition). B, C, Mixed neuron–glia cortical cultures (B) or near pure cortical neuron cultures (C) were exposed for 24 hr to NMDA (12.5 mM) without (white bars) or with ( gray bars) increasing concentration of rhGDNF or MK-801 (10 mM). Medium LDH was assessed at the end of the exposure to excitotoxin (mean 6 SEM; n 5 12 cultures per condition. * indicates significantly different from NMDA; # indicates significantly different from control (sham wash) by ANOVA, followed by Bonferronni–Dunn’s test ( p , 0.05).

Article Snippet: Recombinant human GDNF (rhGDNF) was obtained from R&D Systems (Oxon, UK), and antibodies raised against GDNF, GFRa-1, c-Ret, extracellular signal-regulated kinase 1 (ERK1), phospho-ERKs (p-ERKs), p-c-Jun N-terminal protein kinase (p-JNK), and p-38 were purchased from Santa Cruz (Heidelberg, Germany).

Techniques: Blocking Assay, Concentration Assay, Control

Journal: The Journal of Neuroscience

Article Title: Neuroprotection Mediated by Glial Cell Line-Derived Neurotrophic Factor: Involvement of a Reduction of NMDA-Induced Calcium Influx by the Mitogen-Activated Protein Kinase Pathway

doi: 10.1523/jneurosci.21-09-03024.2001

Figure Lengend Snippet: Figure 6. GDNF does not protect against neuronal death induced by reactive oxygen species. Mixed neuron–glia cortical cultures were ex- posed for 24 hr to FeCl2 (35 mM) without (white bars) or with ( gray bars) 10 ng/ml rhGDNF and with or without Trolox (100 mM). Medium LDH was assessed at the end of FeCl2 exposure (mean 6 SEM; n 5 12 cultures per condition). * indicates significantly different from FeCl2 by ANOVA, followed by Bonferroni–Dunn’s test ( p , 0.05).

Article Snippet: Recombinant human GDNF (rhGDNF) was obtained from R&D Systems (Oxon, UK), and antibodies raised against GDNF, GFRa-1, c-Ret, extracellular signal-regulated kinase 1 (ERK1), phospho-ERKs (p-ERKs), p-c-Jun N-terminal protein kinase (p-JNK), and p-38 were purchased from Santa Cruz (Heidelberg, Germany).

Techniques:

Journal: The Journal of Neuroscience

Article Title: Neuroprotection Mediated by Glial Cell Line-Derived Neurotrophic Factor: Involvement of a Reduction of NMDA-Induced Calcium Influx by the Mitogen-Activated Protein Kinase Pathway

doi: 10.1523/jneurosci.21-09-03024.2001

Figure Lengend Snippet: Figure 5. A GPI-linked protein mediates the neuroprotective activity of rhGDNF. A, Phase-contrast photomicrographs of mixed neuron–glia cortical cultures pretreated or not with PIPLC (0.3 IU/ml for 2 hr at 37°C) after NMDA application (12.5 mM) in the presence or absence of rh- GDNF. Top left, Sham-washed cultures; top right, NMDA-treated cells; bottom left, NMDA-treated cells coincubated with rhGDNF (10 ng/ml); bottom right, cells pretreated with PIPLC and incubated with NMDA and rhGDNF. B, Neuronal death percentage was estimated by LDH release (mean 6 SEM; n 5 12) after a 24 hr exposure to NMDA (12.5 mM) in mixed neuron–glia cortical cultures in the presence ( gray bars) or absence (white bars) of rhGDNF and pretreated or not pretreated with 0.3 IU/ml PIPLC for 2 hr at 37°C. * indicates significantly different from NMDA; # indicates significantly different from NMDA plus GDNF by ANOVA, followed by Bonferroni–Dunn’s test ( p , 0.05).

Article Snippet: Recombinant human GDNF (rhGDNF) was obtained from R&D Systems (Oxon, UK), and antibodies raised against GDNF, GFRa-1, c-Ret, extracellular signal-regulated kinase 1 (ERK1), phospho-ERKs (p-ERKs), p-c-Jun N-terminal protein kinase (p-JNK), and p-38 were purchased from Santa Cruz (Heidelberg, Germany).

Techniques: Activity Assay, Incubation

Journal: The Journal of Neuroscience

Article Title: Neuroprotection Mediated by Glial Cell Line-Derived Neurotrophic Factor: Involvement of a Reduction of NMDA-Induced Calcium Influx by the Mitogen-Activated Protein Kinase Pathway

doi: 10.1523/jneurosci.21-09-03024.2001

Figure Lengend Snippet: Figure 8. The MEK kinase inhibitor U0126 blocks the rhGDNF- induced reduction of NMDA-evoked calcium influx. A, Mouse primary cortical neurons were treated for 15 min with rhGDNF (10 ng/ml) in the presence of U0126 (1 mM) and with or without NMDA (12.5 mM). Aliquots of whole-cell lysates were separated by SDS-PAGE, transferred to membranes, and incubated with the anti-MAPK antibody ( p-ERKs, ERKs). Representative immunoblots are shown, demonstrating the spe- cific blockade of ERKs phosphorylation by U0126 treatment. B, Neuronal death percentage was estimated by LDH release (mean 6 SEM; n 5 12) after a 24 hr exposure to NMDA (12.5 mM) in mixed neuron–glia cortical cultures in the presence ( gray bars) or absence (white bars) of rhGDNF and treated or not treated with 1 mM U0126. * indicates significantly different from NMDA alone; # indicates significantly different from NMDA plus GDNF by ANOVA, followed by Bonferroni–Dunn’s test ( p , 0.05). C, Calcium imaging was performed as described in the legend to Figure 7. Neurons (13–14 DIV) were exposed to serum-free medium for 45 min in the absence (control; vehicle treated; white bars) or presence ( gray bars) of rhGDNF (10 ng/ml) and with (hatched bars) or without (unhatched bars) U0126 (1 mM). Time course of the effect of rhGDNF on the NMDA-induced [Ca 21] increase, with or without treatment with 1 mM of U0126. Data are expressed as an NMDA-induced fold [Ca 21]i. Results represent the mean 6 SEM of three separate preparations (3 coverslips; 20 neurons being imaged per coverslip in a single microscopic field). * indicates significantly different from NMDA; # indicates signif- icantly different from NMDA plus GDNF by ANOVA for repeated measures followed by a Bonferroni–Dunn’s test for multiple comparisons ( p , 0.05).

Article Snippet: Recombinant human GDNF (rhGDNF) was obtained from R&D Systems (Oxon, UK), and antibodies raised against GDNF, GFRa-1, c-Ret, extracellular signal-regulated kinase 1 (ERK1), phospho-ERKs (p-ERKs), p-c-Jun N-terminal protein kinase (p-JNK), and p-38 were purchased from Santa Cruz (Heidelberg, Germany).

Techniques: SDS Page, Incubation, Western Blot, Phospho-proteomics, Imaging, Control

Journal: The Journal of Neuroscience

Article Title: Neuroprotection Mediated by Glial Cell Line-Derived Neurotrophic Factor: Involvement of a Reduction of NMDA-Induced Calcium Influx by the Mitogen-Activated Protein Kinase Pathway

doi: 10.1523/jneurosci.21-09-03024.2001

Figure Lengend Snippet: Figure 7. rhGDNF pretreatment reduces the NMDA-evoked calcium increase in cortical neurons. A, Intracellular free Ca 21 ([Ca 21]i) was measured using fura-2 fluorescence videomicroscopy. Neurons (13–14 DIV) were exposed to a serum-free medium for 45 min in the absence (control, vehicle treated) or presence of rhGDNF (10 ng/ml). Resting [Ca 21 ]i was recorded, and NMDA (100 mM) was applied. [Ca 21]i was measured and analyzed as described in Materials and Methods. B, Rep- resentative graph of NMDA-induced Ca 21 response in neurons pre- treated with rhGDNF ( gray bars) or vehicle (control; white bars) for 45 min. Time course of the effect of rhGDNF on the NMDA-induced [Ca 21] increase, with (hatched bars) or without (unhatched bars) pretreat- ment with 0.3 IU/ml PIPLC for 2 hr. Data are expressed as an NMDA- induced fold increase in [Ca 21]i. Results represent the mean 6 SEM of three separate preparations (3 coverslips; 20 neurons being imaged per coverslip in a single microscopic field). * indicates significantly different from NMDA; # indicates significantly different from NMDA plus GDNF by ANOVA for repeated measures, followed by a Bonferroni–Dunn’s test for multiple comparisons ( p , 0.05). C, Neurons (13–14 DIV) were exposed to serum-free medium containing MK-801 (10 mM) for 45 min in the absence (control; vehicle treated) or presence of rhGDNF (10 ng/ml). Resting [Ca 21 ]i was recorded, and KCl (50 mM) was applied. Represen- tative graph of KCl-induced Ca 21 response in neurons pretreated with rhGDNF or vehicle (control) for 45 min.

Article Snippet: Recombinant human GDNF (rhGDNF) was obtained from R&D Systems (Oxon, UK), and antibodies raised against GDNF, GFRa-1, c-Ret, extracellular signal-regulated kinase 1 (ERK1), phospho-ERKs (p-ERKs), p-c-Jun N-terminal protein kinase (p-JNK), and p-38 were purchased from Santa Cruz (Heidelberg, Germany).

Techniques: Control

Journal: Cell Reports Medicine

Article Title: Autoantibodies against NCAM1 from patients with schizophrenia cause schizophrenia-related behavior and changes in synapses in mice

doi: 10.1016/j.xcrm.2022.100597

Figure Lengend Snippet: Anti-NCAM1 autoantibodies disrupt NCAM1-NCAM1 and NCAM1-GDNF interactions (A) Pull-down assay confirming that IgG purified from a patient with schizophrenia who was positive for anti-NCAM1 autoantibodies disrupts NCAM1-NCAM1 interactions. His-tagged proteins were pulled down by Ni-NTA-agarose, and GST-tagged proteins were pulled down by Glutathione Sepharose. (B) Pull-down assay showing that IgG purified from a patient with schizophrenia who was anti-NCAM1 autoantibody-positive disrupts the NCAM1-GDNF interaction. His-tagged proteins were pulled down by Ni-NTA-agarose, and GST-tagged proteins were pulled down by Glutathione Sepharose.

Article Snippet: GDNF Human Recombinant Protein , Origene , Cat# TP760516.

Techniques: Pull Down Assay, Purification

Journal: Cell Reports Medicine

Article Title: Autoantibodies against NCAM1 from patients with schizophrenia cause schizophrenia-related behavior and changes in synapses in mice

doi: 10.1016/j.xcrm.2022.100597

Figure Lengend Snippet:

Article Snippet: GDNF Human Recombinant Protein , Origene , Cat# TP760516.

Techniques: Virus, Plasmid Preparation, Recombinant, Labeling, cDNA Synthesis, Enzyme-linked Immunosorbent Assay, Software