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: Annals of biomedical engineering

Article Title: Effects of Varied Stimulation Parameters on Adipose-Derived Stem Cell Response to Low-Level Electrical Fields

doi: 10.1007/s10439-021-02875-z

Figure Lengend Snippet: Primer sequences for target genes.

Article Snippet: For the continuous stimulation (i.e., 24 h, 1 Hz regime), a human BDNF ELISA (RayBiotech, ELH-BDNF-2) and a human GDNF ELISA (RayBiotech ELH-GDNF-1) were performed to assess neural-specific factors.

Techniques:

Journal: Annals of biomedical engineering

Article Title: Effects of Varied Stimulation Parameters on Adipose-Derived Stem Cell Response to Low-Level Electrical Fields

doi: 10.1007/s10439-021-02875-z

Figure Lengend Snippet: AC Stimulation: Low-level, intermittent (1 h × 3 days) ES of ASCs. (a) Gene expression results for general wound healing factors VEGF-A and TIMP1 and neural growth factors BDNF and GDNF. (b) VEGF protein levels in secretome media from ASCs stimulated with different pulse frequencies. (c) Secretome from stimulated ASCs was incubated on dermal fibroblasts to determine scratch closure rate (%/h). Data are mean ± std, n = 11–12/group across 3 biological replicates for PCR, n = 3/group biological replicates for ELISA and n = 8 wells/group across 3 biological replicates for wound healing assay. *p value < 0.05, **p value < 0.01, ***p value < 0.0001 compared to NoES.

Article Snippet: For the continuous stimulation (i.e., 24 h, 1 Hz regime), a human BDNF ELISA (RayBiotech, ELH-BDNF-2) and a human GDNF ELISA (RayBiotech ELH-GDNF-1) were performed to assess neural-specific factors.

Techniques: Gene Expression, Incubation, Enzyme-linked Immunosorbent Assay, Wound Healing Assay

Journal: Annals of biomedical engineering

Article Title: Effects of Varied Stimulation Parameters on Adipose-Derived Stem Cell Response to Low-Level Electrical Fields

doi: 10.1007/s10439-021-02875-z

Figure Lengend Snippet: DC Stimulation: ASCs underwent intermittent stimulation (1 h × 3 days) at low field strength. (a) Growth factor gene expression results for ASCs exposed to DC stimulation. (b) ELISA results for VEGF and GDNF in ASC secretome following ES. Data are mean ± std, n = 11–12/group across 3 biological replicates for PCR and n = 3/group biological replicates for ELISA. *p value < 0.05, **p value < 0.01, ***p value < 0.0001 compared to NoES.

Article Snippet: For the continuous stimulation (i.e., 24 h, 1 Hz regime), a human BDNF ELISA (RayBiotech, ELH-BDNF-2) and a human GDNF ELISA (RayBiotech ELH-GDNF-1) were performed to assess neural-specific factors.

Techniques: Gene Expression, Enzyme-linked Immunosorbent Assay

Journal: Annals of biomedical engineering

Article Title: Effects of Varied Stimulation Parameters on Adipose-Derived Stem Cell Response to Low-Level Electrical Fields

doi: 10.1007/s10439-021-02875-z

Figure Lengend Snippet: ASCs were exposed to continuous stimulation (24 h) at 1 Hz pulse frequency. (a) Growth factor gene expression results for ASCs exposed to continuous stimulation. (b) ELISA results for VEGF, BDNF and GDNF in ASC secretome following 24 h of ES. Data are mean ± std, n = 11/group across 3 biological replicates and n = 3/group biological replicates for ELISA. *p value < 0.05, **p value < 0.01, ***p value < 0.0001, #p value = 0.055 compared to NoES.

Article Snippet: For the continuous stimulation (i.e., 24 h, 1 Hz regime), a human BDNF ELISA (RayBiotech, ELH-BDNF-2) and a human GDNF ELISA (RayBiotech ELH-GDNF-1) were performed to assess neural-specific factors.

Techniques: Gene Expression, Enzyme-linked Immunosorbent Assay

Journal: Experimental Neurobiology

Article Title: Glial Cell Line-derived Neurotrophic Factor-overexpressing Human Neural Stem/Progenitor Cells Enhance Therapeutic Efficiency in Rat with Traumatic Spinal Cord Injury

doi: 10.5607/en.2019.28.6.679

Figure Lengend Snippet: Lists of antibodies used for immunostaining and primers used for qPCR

Article Snippet: The levels of human GDNF were measured using a human GDNF DuoSet ELISA Development kit (R&D systems) according to the manufacturer’s instructions.

Techniques: Immunostaining, Immunocytochemistry, Immunohistochemistry, Amplification, Sequencing

Journal: Experimental Neurobiology

Article Title: Glial Cell Line-derived Neurotrophic Factor-overexpressing Human Neural Stem/Progenitor Cells Enhance Therapeutic Efficiency in Rat with Traumatic Spinal Cord Injury

doi: 10.5607/en.2019.28.6.679

Figure Lengend Snippet: In vitro and in vivo characterization of GDNF-hNSPCs. (A) Compared with Mock-hNSPCs (DAPI + , blue), many of GDNF-hNSPCs express GDNF (green). (B) An ELISA of GDNF in Mock- and GDNF-CM. (C, D) Representative images of Mock- and GDNF-hNSPCs stained for differentiation markers and quantification of the percentages of cells that expressed these markers. DAPI + Cells (blue) express nestin (red), GFAP (green), Tuj1 (red), PDGFR (green), and O4 (red). (E, F) Representative images of CM-treated SH-SY5Y cells and quantification of average neurite length under different experimental conditions. (G) hNuc + GDNF–hNSPCs (red) implanted into the injured spinal cord show robust engraftment and an extensive distribution throughout the lesion and adjacent areas. The boxed area is shown at high magnification in the right panel. The asterisk indicates the lesion epicenter and arrows indicate the cell transplantation sites rostral and caudal to the lesion epicenter. (H, I) Representative images of Mock- and GDNF-hNSPCs stained for differentiation markers in vivo and quantification of the percentages of cells that expressed these markers. Confocal images show that hNSPCs positive for the human cell markers Ku80, STEM101, and STEM121 colocalize with differentiation markers (green). (J) Engrafted STEM121 + GDNF-hNSPCs (red) express GDNF (green). Scale bars: 50 μm in A, D; 100 μm in E; 1 mm in G; and 30 μm in I, J. Data represent the means±SEM. *p<0.05 vs. Mock-hNSPCs in C, H; *p<0.05 vs. vehicle, ***p<0.001 vs. vehicle, ##p<0.01 vs. Mock-hNSPCs in F.

Article Snippet: The levels of human GDNF were measured using a human GDNF DuoSet ELISA Development kit (R&D systems) according to the manufacturer’s instructions.

Techniques: In Vitro, In Vivo, Enzyme-linked Immunosorbent Assay, Staining, Transplantation Assay

Journal: Experimental Neurobiology

Article Title: Glial Cell Line-derived Neurotrophic Factor-overexpressing Human Neural Stem/Progenitor Cells Enhance Therapeutic Efficiency in Rat with Traumatic Spinal Cord Injury

doi: 10.5607/en.2019.28.6.679

Figure Lengend Snippet: Grafted GDNF-hNSPCs enhance neurite outgrowth, axonal extension, and myelination in the injured spinal cord. (A) Multiple NF + neuronal processes (green) extend over engrafted hNuc + human cells (red). Compared with Mock-hNSPCs, GDNF-hNSPCs significantly enhance neurite extension and host axonal sprouting. Arrows indicate hNuc + donor-derived cells (red) and arrowheads indicate NF + neuronal fibers (green). (B) Under high magnification, many NF + neuronal processes (red) are co-labeled with an anti-MBP antibody (green). Arrowheads indicate co-labeled myelinated neuronal fibers (yellow or orange). (C) The intensity of MBP staining in engrafted and adjacent areas is markedly higher in the GDNF-hNSPC-treated group than in the Mock-hNSPC-treated group. Arrows indicate hNuc + donor-derived cells (red) and arrowheads indicate MBP + myelinated neuronal fibers (green). (D) Representative images of LFB staining (blue) in the lesion and adjacent areas of the spinal cord. Dashed lines indicate demyelinated areas. (E) Quantification of the demyelinated volume in the different experimental groups. (F, G) Representative electron microscopic images of axially sectioned spinal cords (T 8 ~T 10 ) and quantification of G-ratio. (H, I) Representative images of Schwann/2E immunostaining (green) in the injured spinal cords and quantification of immunostaining density in the different experimental groups. Scale bars: 1 mm in A, C, D; 100 μm in B; 1 μm in F, and 500 μm in H. Data represent the means±SEM. *p<0.05 vs. vehicle, #p<0.01 vs. Mock-hNSPCs.

Article Snippet: The levels of human GDNF were measured using a human GDNF DuoSet ELISA Development kit (R&D systems) according to the manufacturer’s instructions.

Techniques: Derivative Assay, Labeling, Staining, Immunostaining

Journal: Experimental Neurobiology

Article Title: Glial Cell Line-derived Neurotrophic Factor-overexpressing Human Neural Stem/Progenitor Cells Enhance Therapeutic Efficiency in Rat with Traumatic Spinal Cord Injury

doi: 10.5607/en.2019.28.6.679

Figure Lengend Snippet: Grafted GDNF-hNSPCs protect the dCST, promote the formation of detour circuits, and reduce lesion volume and glia scar formation. (A) Representative images of BDA-labeled dCST fibers (red) at 6 mm rostral to the lesion epicenter in the different experimental groups. The boxed areas in the left panels are shown at high magnification in the right panels. (B) Quantification of the number of dCST fibers up to 6 mm rostral and caudal to the lesion epicenter. (C) Representative images of dCST collaterals (black, left panel) and FG-labeled PSNs (white, right panel) in the cervical enlargement of the spinal cord in the different experimental groups. The white dashed lines in the right panel indicate the margins between gray and white matter of the spinal cord. (D) Quantification of dCST collaterals and FG-labeled PSNs in the cervical enlargement. (E) Representative images of the contacts between dCST collaterals (black) and PSNs (brown) in the cervical enlargement. Arrowheads show closely located collateral fibers and PSNs. (F) Quantification of the number of contacts between dCST collaterals and PSNs. (G) Representative images of GFAP immunostaining in the lesion and adjacent areas of the spinal cord in the different experimental groups. Dashed lines indicate GFAP-negative areas. (H) Quantification of lesion volume, spared tissue volume, and glial scars. Scale bars: 100 μm in A, C; 40 μm in E; and 1 mm in G. Data represent the means±SEM. *p<0.05 vs. vehicle, ***p<0.001 vs. vehicle, #p<0.01 vs. Mock-hNSPCs.

Article Snippet: The levels of human GDNF were measured using a human GDNF DuoSet ELISA Development kit (R&D systems) according to the manufacturer’s instructions.

Techniques: Labeling, Immunostaining

Journal: Experimental Neurobiology

Article Title: Glial Cell Line-derived Neurotrophic Factor-overexpressing Human Neural Stem/Progenitor Cells Enhance Therapeutic Efficiency in Rat with Traumatic Spinal Cord Injury

doi: 10.5607/en.2019.28.6.679

Figure Lengend Snippet: Transplantation of GDNF-hNSPCs promotes motosensory recovery, reduces expression of voltage-gated sodium channels and NPY, and increases expression of GABA. (A) The BBB open-field walking scores before transplantation and at various time points post-transplantation. (B) Von Frey tests for mechanical allodynia in lesioned hindlimbs of rats with SCI before transplantation and at various time points post-transplantation. (C) qRT-PCR analysis of Nav1.3 and Nav1.9 expression in the T 8 ~T 10 segment of the spinal cord. (D, E) Representative images and quantification of NPY expression in lamina I of the dorsal horn in the T 8 segment of the spinal cord. (F, G) Representative images and quantification of GABA expression in the T 8 ~T 10 segment of the spinal cord. (H) Many engrafted hNuc + GDNF-hNSPCs (red, arrowheads) express GABA (green, arrowheads) adjacent endogenous GABA + cells (green, arrows). Scale bars: 200 μm in D; 500 μm in F; and 50 μm in H. Data represent the means±SEM. *p<0.05 vs. vehicle, **p<0.01 vs. vehicle, ***p<0.001 vs. vehicle, #p<0.05 vs. Mock-hNSPCs, ##p<0.01 vs. Mock-hNSPCs in A, B. *p<0.05 vs. vehicle, #p<0.05 vs. Mock-hNSPCs in C, E, and G.

Article Snippet: The levels of human GDNF were measured using a human GDNF DuoSet ELISA Development kit (R&D systems) according to the manufacturer’s instructions.

Techniques: Transplantation Assay, Expressing, Quantitative RT-PCR

Journal: Current Research in Pharmacology and Drug Discovery

Article Title: Long-term, stable, targeted biodelivery and efficacy of GDNF from encapsulated cells in the rat and Goettingen miniature pig brain

doi: 10.1016/j.crphar.2020.04.001

Figure Lengend Snippet: (a) ELISA analysis of encapsulated clonal ARPE-19 ​cell lines demonstrates robust continued secretion of GDNF from clone-125. While GDNF secretion from clones 20 and 120 decreased between 4 and 8 weeks, secretion from clone-125 remained stable and even rose during the same period in vivo. (b) Immunohistochemistry of tissue sections at 8 weeks post implant confirms robust diffusion of GDNF from encapsulated clone-125 ​cells throughput the implanted striatum and overlying cortex. Scale bar, 500 ​μm in panel b and 75 ​μm in panel c. H&E staining on longitudinal sections of devices confirms excellent viability of encapsulated clone −125 ​cells. Clone-125 was used for all subsequent studies. Data are expressed as mean ​± ​SEM of 4 animals each with bilateral implants per group/time-point. ∗∗∗p<0.0001; Student's t test for unpaired data.

Article Snippet: A commercially available GDNF ELISA kit (DuoSet® for human GDNF R&D Systems, Minneapolis, MN) was used to quantify GDNF device secretion prior to implantation and again following retrieval from the brain.

Techniques: Enzyme-linked Immunosorbent Assay, Clone Assay, In Vivo, Immunohistochemistry, Diffusion-based Assay, Staining

Journal: Current Research in Pharmacology and Drug Discovery

Article Title: Long-term, stable, targeted biodelivery and efficacy of GDNF from encapsulated cells in the rat and Goettingen miniature pig brain

doi: 10.1016/j.crphar.2020.04.001

Figure Lengend Snippet: Long-term, sustained delivery of GDNF and resulting elevation tissue levels of GDNF within the implanted striatum. Fig. 2 a demonstrates that GDNF device secretion peaks early post implantation but then stabilizes and remains consistent for at least 6 months in vivo. Fig. 2 b confirms that GDNF delivery results in sustained tissue levels within the implanted striatum. When explanted from the striatum the output of the devices over time generally paralleled the enhanced tissue concentrations with an early peak in delivery followed by a longer, sustained output similar to that noted from the same devices immediately prior to implantation. All data are expressed as mean ​± ​SEM of 4 animals each with bilateral implants per time-point. BLQ ​= ​below limit of quantitation.

Article Snippet: A commercially available GDNF ELISA kit (DuoSet® for human GDNF R&D Systems, Minneapolis, MN) was used to quantify GDNF device secretion prior to implantation and again following retrieval from the brain.

Techniques: In Vivo, Quantitation Assay

Journal: Current Research in Pharmacology and Drug Discovery

Article Title: Long-term, stable, targeted biodelivery and efficacy of GDNF from encapsulated cells in the rat and Goettingen miniature pig brain

doi: 10.1016/j.crphar.2020.04.001

Figure Lengend Snippet: GDNF secretion from encapsulated cells implanted into intact versus 6-OHDALesioned.

Article Snippet: A commercially available GDNF ELISA kit (DuoSet® for human GDNF R&D Systems, Minneapolis, MN) was used to quantify GDNF device secretion prior to implantation and again following retrieval from the brain.

Techniques:

Journal: Current Research in Pharmacology and Drug Discovery

Article Title: Long-term, stable, targeted biodelivery and efficacy of GDNF from encapsulated cells in the rat and Goettingen miniature pig brain

doi: 10.1016/j.crphar.2020.04.001

Figure Lengend Snippet: Photomicrographs of substantia nigra stained for TH immunoreactivity illustrating loss of dopaminergic neurons following 6-OHDA and preservation of those neurons following GDNF treatment (a). Quantitation of dopaminergic neurons in the substantia nigra demonstrates virtually complete protection of lesioned neurons (b). Data are presented as mean ​± ​SEM % of TH-positive neurons relative to the intact, non-lesioned hemisphere of 8 animals per group. Scale bars, 200 ​μm. Multiple comparisons were based on significant main effects of treatment resulting from the ANOVA described in Results: ∗∗p<0.01.

Article Snippet: A commercially available GDNF ELISA kit (DuoSet® for human GDNF R&D Systems, Minneapolis, MN) was used to quantify GDNF device secretion prior to implantation and again following retrieval from the brain.

Techniques: Staining, Preserving, Quantitation Assay

Journal: Current Research in Pharmacology and Drug Discovery

Article Title: Long-term, stable, targeted biodelivery and efficacy of GDNF from encapsulated cells in the rat and Goettingen miniature pig brain

doi: 10.1016/j.crphar.2020.04.001

Figure Lengend Snippet: Photomicrographs of striatum stained for TH immunoreactivity (a) and quantitation of TH fiber density (b) following 6-OHDA with and without GDNF treatment illustrates a robust ability of GDNF to preserve dopaminergic fibers. Scale bars ​= ​500 ​μm Data are presented as mean ​± ​SEM % of TH-positive fibers relative to the intact, non-lesioned hemisphere of 8 animals per group. Multiple comparisons were based on significant main effects of treatment resulting from the ANOVA described in Results: ∗∗p<0.01.

Article Snippet: A commercially available GDNF ELISA kit (DuoSet® for human GDNF R&D Systems, Minneapolis, MN) was used to quantify GDNF device secretion prior to implantation and again following retrieval from the brain.

Techniques: Staining, Quantitation Assay

Journal: Current Research in Pharmacology and Drug Discovery

Article Title: Long-term, stable, targeted biodelivery and efficacy of GDNF from encapsulated cells in the rat and Goettingen miniature pig brain

doi: 10.1016/j.crphar.2020.04.001

Figure Lengend Snippet: Normal weight gain and neurological performance is maintained in lesioned animals by implantation of GDNF devices. (a) Body weights of animals with and without GDNF treatment. (b–d) Performance in the cylinder, placing, and stepping tests are all significantly impaired at 2 and 4 weeks following intrastriatal injections of 6-OHDA. In contrast, performance on each of these tests is preserved by GNDF implants with treated animals performing comparably to pre-surgery levels (pre-implantation and pre-lesion). Data are presented as mean ​± ​SEM of 8 animals per group. Multiple comparisons were based on significant main effects or interactions resulting from ANOVAs that are described in Results: ∗∗p<0.01.

Article Snippet: A commercially available GDNF ELISA kit (DuoSet® for human GDNF R&D Systems, Minneapolis, MN) was used to quantify GDNF device secretion prior to implantation and again following retrieval from the brain.

Techniques:

Journal: Current Research in Pharmacology and Drug Discovery

Article Title: Long-term, stable, targeted biodelivery and efficacy of GDNF from encapsulated cells in the rat and Goettingen miniature pig brain

doi: 10.1016/j.crphar.2020.04.001

Figure Lengend Snippet: Long-term, normal weight gain (a) and neurological performance in rats receiving GDNF implants 1 month post 6-OHDA. Then evaluated for 62 weeks. Testing on both the cylinder and placing tests (b-c) revealed a smooth and ever growing improvement in performance over the 62 week testing period. Data are presented as mean ​± ​SEM of 8 animals per group. Multiple comparisons were based on significant main effects or interactions resulting from ANOVAs that are described in Results: ∗p<0.05.

Article Snippet: A commercially available GDNF ELISA kit (DuoSet® for human GDNF R&D Systems, Minneapolis, MN) was used to quantify GDNF device secretion prior to implantation and again following retrieval from the brain.

Techniques:

Journal: Current Research in Pharmacology and Drug Discovery

Article Title: Long-term, stable, targeted biodelivery and efficacy of GDNF from encapsulated cells in the rat and Goettingen miniature pig brain

doi: 10.1016/j.crphar.2020.04.001

Figure Lengend Snippet: GDNF immunoreactivity in rat striatum 62 weeks following implantation of encapsulated GDNF-secreting cells (a) is associated with a pronounced preservation of TH-positive fibers that normally occurs post 6-OHDA (b–c). Scale bars ​= ​500 ​μm.

Article Snippet: A commercially available GDNF ELISA kit (DuoSet® for human GDNF R&D Systems, Minneapolis, MN) was used to quantify GDNF device secretion prior to implantation and again following retrieval from the brain.

Techniques: Preserving

Journal: Current Research in Pharmacology and Drug Discovery

Article Title: Long-term, stable, targeted biodelivery and efficacy of GDNF from encapsulated cells in the rat and Goettingen miniature pig brain

doi: 10.1016/j.crphar.2020.04.001

Figure Lengend Snippet: Implantation of 2 clinical-sized devices into the right putamen of minipigs delivers GDNF for 12 weeks and produces widespread delivery of GDNF (a–b) throughout the implanted striatum. GDNF delivery is also associated with a profound biological upregulation on dopaminergic function as determined by enhanced TH-immunoreactivity and quantitative optical densitometry in the striatum (c–d). Scale bars ​= ​5 ​mm. Data are presented as mean ​± ​SEM of 6 animals. ∗p<0.05; ∗∗p<0.01, Student's t test.

Article Snippet: A commercially available GDNF ELISA kit (DuoSet® for human GDNF R&D Systems, Minneapolis, MN) was used to quantify GDNF device secretion prior to implantation and again following retrieval from the brain.

Techniques:

Journal: Current Research in Pharmacology and Drug Discovery

Article Title: Long-term, stable, targeted biodelivery and efficacy of GDNF from encapsulated cells in the rat and Goettingen miniature pig brain

doi: 10.1016/j.crphar.2020.04.001

Figure Lengend Snippet: Serum and CSF measurements following GDNF implants in minipig Brain.

Article Snippet: A commercially available GDNF ELISA kit (DuoSet® for human GDNF R&D Systems, Minneapolis, MN) was used to quantify GDNF device secretion prior to implantation and again following retrieval from the brain.

Techniques:

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Destabilizing Domains Enable Long-Term and Inert Regulation of GDNF Expression in the Brain

doi: 10.1016/j.omtm.2018.08.008

Figure Lengend Snippet: GDNF-F-DD Can Be Regulated in a Dose-Dependent Manner, Revert Rapidly to Basal Levels, and Be Induced Multiple Times In Vivo without Loss of Efficiency LVs expressing GDNF-F-DD were delivered to the striatum of animals. One group of animals was given LV expressing GDNF, and another group of animals was untransduced (UT). After 2 days, the animals were given 0.05, 0.1, 0.2, or 0.5 mg/mL TMP continuously in the drinking water for 3 weeks. (A) GDNF ELISA was performed on striatum samples. (B) GDNF ELISA was performed in substantia nigra samples. A one-way ANOVA with Dunnett multiple comparison tests was performed (n = 4–6 per group). To determine the kinetics of the DD system in vivo , LVs expressing GDNF-F-DD were delivered to the right striatum of SD rats. The animals were given TMP (ON) for up to 5 weeks, and then the TMP was withdrawn and the animals given regular drinking water (OFF) for another 5 weeks. (C) Histological analysis and quantification of pRPS6 neurons were performed to determine how fast the DD system could be turned on and off (n = 3–5 per group). A similar experimental setup was used to determine whether the DD system could be induced multiple times. After LV GDNF-F-DD delivery, animals were divided into five groups. The first group was ON for 4 weeks (first induction). The second was ON for 4 weeks and OFF for 3 weeks. The next group of animals was induced 2 times, ON-OFF-ON (second induction). The following group was ON-OFF-ON-OFF. The last group was induced 3 times, ON-OFF-ON-OFF-ON (third induction). (D) Quantification of activated SNpc cells was performed in sections stained for pRPS6 (n = 4–5 per group). (E) Analysis of GDNF-F-DD expression was done using striatal sections stained for GDNF (n = 4–5 per group). The x axis indicates weeks after LV delivery (see also Figure S1 ). A one-way ANOVA with Dunnett multiple comparison tests was performed. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. ns, not significant. Error bars represent mean ± SEM or mean + SEM.

Article Snippet: The following primary antibodies were used: rabbit anti-pRPS6 (#2211, 1:400, Cell Signaling Technology, Danvers, MA, USA), goat anti-human GDNF (AF-212-NA, 1:1,000, R&D Systems Europe), rabbit anti-DHFR DD (1:20,000, a kind gift from the Wandless lab that has been used previously ), mouse anti-RFP (ab65856, 1:1,000, Abcam, Cambridge, UK), mouse anti-NeuN (MAB377, 1:1,000, Merck Millipore, Darmstadt, Germany), rat anti-CD11B (MCA275R, 1:1,000, Bio-Rad Laboratories, Solna, Sweden), and mouse anti-CD8 alpha (ab33786, 1:1,000, Abcam, Cambridge, UK).

Techniques: In Vivo, Expressing, Enzyme-linked Immunosorbent Assay, Comparison, Staining

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Destabilizing Domains Enable Long-Term and Inert Regulation of GDNF Expression in the Brain

doi: 10.1016/j.omtm.2018.08.008

Figure Lengend Snippet: Experimental Design and LV Used to Determine Safety of the DD System (A) To determine the safety of DD induction in vivo , animals were injected in the striatum with LV expressing MRFP or YFP-DD and LV expressing GDNF-F-DD and MRFP from two separate expression assets (GDNF-F-DD/MRFP). Half of the GDNF-F-DD/MRFP animals were given regular draining water (OFF), and the other half were given TMP (ON). The last group of animals was injected with processed media from cells transduced only with MRFP transfer plasmid (Sham LV). Twelve or 24 weeks after LV delivery, sera were taken for DD/MRFP ELISA, animals were perfused, and their brains were processed for histology. Expression of the system was assayed using immunohistochemistry for DD, GDNF, and MRFP. Toxicity was assessed using immunohistochemistry for NeuN, CD11B, and CD8. (B) Detailed maps of the transfer vectors used for the experiment.

Article Snippet: The following primary antibodies were used: rabbit anti-pRPS6 (#2211, 1:400, Cell Signaling Technology, Danvers, MA, USA), goat anti-human GDNF (AF-212-NA, 1:1,000, R&D Systems Europe), rabbit anti-DHFR DD (1:20,000, a kind gift from the Wandless lab that has been used previously ), mouse anti-RFP (ab65856, 1:1,000, Abcam, Cambridge, UK), mouse anti-NeuN (MAB377, 1:1,000, Merck Millipore, Darmstadt, Germany), rat anti-CD11B (MCA275R, 1:1,000, Bio-Rad Laboratories, Solna, Sweden), and mouse anti-CD8 alpha (ab33786, 1:1,000, Abcam, Cambridge, UK).

Techniques: In Vivo, Injection, Expressing, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Immunohistochemistry

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Destabilizing Domains Enable Long-Term and Inert Regulation of GDNF Expression in the Brain

doi: 10.1016/j.omtm.2018.08.008

Figure Lengend Snippet: Lack of Neurotoxicity during Long-Term GDNF-F-DD Expression in the Brain

Article Snippet: The following primary antibodies were used: rabbit anti-pRPS6 (#2211, 1:400, Cell Signaling Technology, Danvers, MA, USA), goat anti-human GDNF (AF-212-NA, 1:1,000, R&D Systems Europe), rabbit anti-DHFR DD (1:20,000, a kind gift from the Wandless lab that has been used previously ), mouse anti-RFP (ab65856, 1:1,000, Abcam, Cambridge, UK), mouse anti-NeuN (MAB377, 1:1,000, Merck Millipore, Darmstadt, Germany), rat anti-CD11B (MCA275R, 1:1,000, Bio-Rad Laboratories, Solna, Sweden), and mouse anti-CD8 alpha (ab33786, 1:1,000, Abcam, Cambridge, UK).

Techniques: Expressing