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: Plasmid Preparation, Recombinant, Labeling, Enzyme-linked Immunosorbent Assay, Software

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Phosphorylation of protocadherin proteins by the receptor tyrosine kinase Ret

doi: 10.1073/pnas.1007182107

Figure Lengend Snippet: Ret regulates phosphorylation and protein levels of Pcdhα and Pcdhγ. (A) Differentiated CAD cells expressing Pcdhα4-TAP (α4-TAP), Pcdhγb7-TAP (γb7-TAP), or empty TAP vector (TAP) were left untreated or stimulated with GDNF/GFRα1 for 0.5 or 3 h. Anti-FLAG (TAP) immunoprecipitates (IPs) were blotted with anti-phosphotyrosine (anti-P-Tyr). The blot was reprobed with an anti-HA (TAP) antibody. Total cell lysates were blotted with anti-MAPK. (B) CAD cells expressing Pcdhα4-ΔC3-TAP (ΔC3-TAP), Pcdhα4-TAP, Pcdhγb7-TAP, or empty TAP vector were infected with anti-Ret shRNA or control anti-GFP shRNA lentivirus. Anti-FLAG (TAP) IPs of differentiated cells were blotted for P-Tyr, and the blot was reprobed with an anti-HA (TAP) antibody. Total lysate was blotted with an anti-Ret antibody. (C) Total lysate of differentiated CAD cells expressing lentiviral shRNA plasmids targeting Ret or GFP was blotted for Ret9 and Ret51, Pcdhα, Pcdhγ, and TuJ1.

Article Snippet: Cells were induced with mouse recombinant GDNF (R&D Systems) and recombinant GDNF receptor (GFR)α1/Fc fusion (R&D Systems) as indicated.

Techniques: Phospho-proteomics, Expressing, Plasmid Preparation, Infection, shRNA, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Phosphorylation of protocadherin proteins by the receptor tyrosine kinase Ret

doi: 10.1073/pnas.1007182107

Figure Lengend Snippet: Pcdhγ undergoes GDNF-induced phosphorylation and interacts with Ret in MNs and sympathetic neurons. (A) Anti-Pcdhγ or control rabbit serum (IgG) immunoprecipitates (IPs) of MN lysate were blotted with anti-Ret and anti-P-Tyr antibodies. The blot was reprobed with an anti-Pcdhγ antibody. (B) MNs were left untreated, stimulated for 30 min with GDNF/GFRα1, or treated for 20 min with PP2 prior to 30 min of GDNF/GFRα1 stimulation. Anti-Pcdhγ or rabbit serum control (IgG) IPs were blotted with anti-P-Tyr and anti-Pcdhγ. Total cell lysate was blotted with anti-Ret, anti-P-MAPK, and anti-MAPK. (C) Sympathetic neurons were left untreated or stimulated for 30 min with GDNF. Anti-Pcdhα and anti-Pcdhγ IPs were blotted with anti-P-Tyr and anti-pan-Ret. The blots were reprobed with anti-Pcdhα and anti-Pcdhγ antibody. Total cell lysate was blotted with anti-Ret and anti-Tuj1. (D) Cortical glia were left untreated, stimulated with GDNF/GFRα1 for 30 min, or treated with PP2 before 30 min of GDNF/GFRα1 stimulation. Anti-Pcdhγ or rabbit control serum (IgG) IPs were blotted with anti-P-Tyr antibody. The blot was reprobed with an anti-Pcdhγ antibody.

Article Snippet: Cells were induced with mouse recombinant GDNF (R&D Systems) and recombinant GDNF receptor (GFR)α1/Fc fusion (R&D Systems) as indicated.

Techniques: Phospho-proteomics, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Phosphorylation of protocadherin proteins by the receptor tyrosine kinase Ret

doi: 10.1073/pnas.1007182107

Figure Lengend Snippet: Pcdhs are required for stabilization of activated Ret. (A) Differentiated CAD cells stably expressing Pcdhα4-TAP (α4-TAP), Pcdhγb7-TAP (γb7-TAP), or TAP vector control (TAP) were left untreated or treated with GDNF/GFRα1 for 0.5 or 3 h. Anti-Ret51 and anti-FLAG (TAP) immunoprecipitates (IPs) were blotted with anti-Ret51 and anti-P-Ret. The blots were reprobed with anti-Ret51 antibody. The anti-FLAG (TAP) immunoprecipitation blot was reprobed with anti-HA antibody. (B) CAD cells infected with anti-Pcdhα, anti-Pcdhγ, anti-GFP, or anti-Ret lentiviral shRNA were differentiated. Total cell lysate was blotted for Ret9, Ret51, Pcdhα, Pcdhγ, and Tuj1. (C) Sympathetic neurons were infected with lentivirus encoding shRNA plasmids targeting Pcdhα and Pcdhγ or GFP. Neurons were cultured for an additional 7 d and stimulated with GDNF for various times. Anti-Ret51 IPs were blotted with anti-Ret51 and anti-P-Ret. Antiubiquitin IPs were blotted with anti-Ret51. Total cell lysate was blotted with anti-P-Ret, anti-Ret51, Pcdhα, Pcdhγ, and Tuj1 antibodies.

Article Snippet: Cells were induced with mouse recombinant GDNF (R&D Systems) and recombinant GDNF receptor (GFR)α1/Fc fusion (R&D Systems) as indicated.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Control, Immunoprecipitation, Infection, shRNA, Cell Culture

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Phosphorylation of protocadherin proteins by the receptor tyrosine kinase Ret

doi: 10.1073/pnas.1007182107

Figure Lengend Snippet: Model of Pcdh and Ret interaction and stabilization in CAD and sympathetic neurons. Activation of Ret with GDNF/GFRα1 leads to ubiquitination and rapid degradation. Pcdhs not bound to Ret also undergo degradation. Activated Ret bound to Pcdh is stabilized. Pcdhs bound to activated Ret are stabilized and phosphorylated and might initiate downstream signaling.

Article Snippet: Cells were induced with mouse recombinant GDNF (R&D Systems) and recombinant GDNF receptor (GFR)α1/Fc fusion (R&D Systems) as indicated.

Techniques: Activation Assay, Ubiquitin Proteomics

Journal:

Article Title: Nucleus Accumbens-Derived GDNF is a Retrograde Enhancer of Dopaminergic Tone in the Mesocorticolimbic System

doi: 10.1523/JNEUROSCI.3909-10.2010

Figure Lengend Snippet: GDNF (10 μg/2μl) and/or vehicle were bilaterally (A–C) or unilaterally (D) infused into the NAc, and VTA slices were prepared 12 hrs later (the time point was chosen based on a previous study (Tomac et al., 1995b)). A, In vivo application of GDNF in the NAc elicits an increase in the firing rate of VTA neurons. Cumulative probability plot comparing spontaneous firing rates of individual neurons in VTA slices from vehicle- (black circles) and GDNF- (red circles) treated rats. ** p < 0.01 vs. vehicle by Kolmogorov-Smirnov test, n = 19 cells from 3 rats for each group. B,C, NAc-derived GDNF increases excitatory but decreases inhibitory synaptic drives to VTA neurons. B, Sample traces of mEPSCs (top) and mIPSCs (bottom) after intra-NAc infusion of vehicle or GDNF. Scale bars: 0.2 sec and 15 pA (mEPSCs); 0.3 sec and 60 pA (mIPSCs). C, Bar graphs summarizing the mean frequencies (top) and amplitudes (bottom) of mEPSCs and mIPSCs. n = 11 (mEPSCs, vehicle), 12 (mEPSCs, GDNF), 15 (mIPSCs, vehicle), and 16 (mIPSCs, GDNF) slices. * p < 0.05, ** p < 0.01 t-test. D, Intra-NAc infusion of GDNF into the NAc leads to ERK1/2 in VTA DA neurons. Images show dual channel immunofluorescence for phospho-ERK1/2 (p-ERK1/2, Red), TH (Green), and overlay (Yellow). Images are representative of results from two rats. Scale bars, 500 μm (Left) and 50 μm (Right). Histological verification of placement of GDNF and vehicle infusions into the NAc is shown in Figure S2.

Article Snippet: Recombinant human GDNF was obtained from R&D System (Minneapolis, MN).

Techniques: In Vivo, Derivative Assay, Immunofluorescence

Journal:

Article Title: Nucleus Accumbens-Derived GDNF is a Retrograde Enhancer of Dopaminergic Tone in the Mesocorticolimbic System

doi: 10.1523/JNEUROSCI.3909-10.2010

Figure Lengend Snippet: The retrograde tracer, Neuro-DiI, was injected bilaterally into the NAc. A, A representative coronal section confirming the injection sites within the NAc. Arrowheads indicate tracer deposit. The double arrowheads indicate the anterior commissure. Scale bar, 1 mm. B, Representative images showing that numerous Neuro-DiI-labeled VTA neurons are also TH-positive. Shown are dual-channel fluorescent images for DiI (Red), TH (Green), and overlay (Yellow). The arrowheads indicate cells labeled with DiI and stained for TH (Right). Scale bars, 500 μm (Left) and 50 μm (Right). C, Representative images showing a DiI-labeled VTA neuron that was selected for electrophysiology. Upper panel, Red fluorescent image. Bottom panel, DIC image. Scale bar, 20 μm. D, A bar graph summarizing the mean increase by GDNF (200 ng/ml) in the firing rate of Neuro-DiI labeled VTA neurons. *p < 0.05. n = 8 cells.

Article Snippet: Recombinant human GDNF was obtained from R&D System (Minneapolis, MN).

Techniques: Injection, Labeling, Staining

Journal:

Article Title: Nucleus Accumbens-Derived GDNF is a Retrograde Enhancer of Dopaminergic Tone in the Mesocorticolimbic System

doi: 10.1523/JNEUROSCI.3909-10.2010

Figure Lengend Snippet: A, Left, Time course of dialysate concentrations of DA from the NAc before and after intra-VTA infusion of 10 μg/μl/side (black circles) or vehicle (Veh, white circles). Two- way ANOVA (Treatment × Fractions) shows a significant effect of Treatment (F(1, 156) = 9.40, p < 0.01) and Fraction (F(13, 159) = 4.88, p < 0.001), and a significant interaction between both factors (F(13, 159) = 2.75, p < 0.01). Post-hoc analysis using the method of contrasts shows a significant difference between the vehicle and the GDNF conditions from fractions 5 to 14 (Ts > 1.81, ps < 0.05). The basal concentration of DA in dialysate was 1.97 ± 0.53 and 2.14 ± 0.42 nM for the Veh and GDNF group, respectively. Right, Bar graph comparing the DA levels in the NAc following GDNF or vehicle injections into the VTA. The values were averaged from fractions 5 to 8. n = 8 (Veh), n = 6 (GDNF). B, Left, The MEK inhibitor U0126 (0.5 μg/μl/side) or vehicle were infused in the VTA 1 hr before the application of GDNF (10 μg/μl/side) or vehicle. Time course of dialysate concentrations of DA from the NAc before and after intra-VTA infusion of, GDNF (black circles) or GDNF/U0126 (white circles). Two-way ANOVA (Treatment × Fractions) shows a significant effect of Treatment (F(1, 156) = 8.35, p < 0.01) and Fraction (F(13, 159) = 4.03, p < 0.001), and no interaction between both factors (F(13, 159) = 1.86, p = 0.12). Post-hoc analysis using the method of contrasts shows a significant difference between the Veh/GDNF and the U0126/GDNF conditions from fractions 11, 12, 15 and 17 (ts > 1.68, ps < 0.05). The basal concentration of DA in dialysate was 0.75 ± 0.18 and 1.13 ± 0.15 nM for the Veh/GDNF and U0126/GDNF group, respectively. Right, Bar graph comparing the DA levels in the NAc following vehicle, U0126 or GDNF injections into the VTA. The values were averaged from fractions 5 to 8 for the vehicle and U0126 injections and 9 to 12 for the GDNF injections. n = 6 (Veh/GDNF), n = 8 (U0129/GDNF). A&B, Intra-VTA infusion of 75 ng of baclofen confirmed the functional connection between the VTA and NAc placements by reducing NAc DA overflow in the four groups. * p < 0.05, ** p < 0.01.

Article Snippet: Recombinant human GDNF was obtained from R&D System (Minneapolis, MN).

Techniques: Concentration Assay, Functional Assay

Journal:

Article Title: Nucleus Accumbens-Derived GDNF is a Retrograde Enhancer of Dopaminergic Tone in the Mesocorticolimbic System

doi: 10.1523/JNEUROSCI.3909-10.2010

Figure Lengend Snippet: Recombinant adeno-virus containing GDNF shRNA (Adv-shGDNF) or scrambled RNA (Adv-SCR) were bilaterally infused into the NAc of rats. A, Viral infection in the NAc was confirmed by GFP fluorescence 18 days post-injection (D18). The white boxes in a and b indicate the position of the medium spiny neuron shown in b and the position of the spiny dendrite shown in c–d (c and d are the same dendrite in color (c) and black and white (d), respectively). Scale bar, 2 mm (a), 50 μm (b), and 10 μm (c). B, Adv-shGDNF decreases GDNF expression in the NAc. The NAc tissue from Adv-shGDNF and Adv-SCR infected rats was dissected at D18 for RT-PCR analysis of mRNA levels of GDNF. Bar graph depicts the average GDNF/GAPDH ratio. * p < 0.05. n = 5 rats for each group. C, Downregulation of GDNF in the NAc does not alter the frequency of VTA neuronal firing evoked by somatic current injections. Cell membrane potentials were brought to −60 mV in whole-cell current-clamp mode, and a depolarization step of 120 pA (0.5 sec) was injected to induce evoked firing. Left, Sample voltage traces response to the current injection in slices from Adv-SCR- (SCR, top) and Adv-shGDNF- (shGDNF, bottom) treated rats. Scale, 30 mV, 100 ms. Middle and Right, bar graphs depicting no difference in firing rate (Middle) and latency (Right) of evoked VTA neuronal firing between Adv-SCR- and Adv-shGDNF-treated rats. The frequency was measured between the first 2 spikes. n = 35 (SCR) and 34 (shGDNF) neurons from 13 rats for each group. The latency was defined as the duration between the onset of current injection to the peak of the first spike. n = 36 (SCR) and 35 (shGDNF) neurons from 13 rats for each group. D, Downregulation of GDNF in the NAc decreases the spontaneous firing rate of VTA neurons. Left, Representative traces of spontaneous firing of VTA neurons in Adv-SCR-(SCR, top) and Adv-shGDNF- (shGDNF, bottom) infected rats. Note that the inter-spike interval is larger (thus the frequency is lower) in the bottom trace than in the top trace. Right, Cumulative probability plot comparing individual neurons in slices from Adv-shGDNF- and Adv-SCR-treated rats. * p < 0.05 by Kolmogorov-Smirnov test, n = 33 neurons from 9 rats for each group. See also Figure S1.

Article Snippet: Recombinant human GDNF was obtained from R&D System (Minneapolis, MN).

Techniques: Recombinant, Virus, shRNA, Infection, Fluorescence, Injection, Expressing, Reverse Transcription Polymerase Chain Reaction, Membrane

Journal:

Article Title: Nucleus Accumbens-Derived GDNF is a Retrograde Enhancer of Dopaminergic Tone in the Mesocorticolimbic System

doi: 10.1523/JNEUROSCI.3909-10.2010

Figure Lengend Snippet: A, Intra-VTA infusion of GDNF produces an increase in the spontaneous firing rate of VTA neurons. GDNF (10 μg/μl) or vehicle was bilaterally infused into the VTA, and slices were prepared 10 min later. Cumulative probability plot was constructed to compare the firing rates of individual neurons in slices from vehicle- and GDNF-treated rats. ** p < 0.01 vs. vehicle by Kolmogorov-Smirnov test, n = 12 cells from 3 rats for each group. B–D, Bath-applied GDNF increases the spontaneous firing frequency of DA neurons. B, Sample traces of spontaneous firing in a tight-seal cell-attached recording before (Baseline) and during (14 min) bath application of GDNF (200 ng/ml). Scale bar, 0.5 sec. C, Averaged time course showing that bath application of GDNF (200 ng/ml, black circles) (n = 14), but not its heat-inactivated form (heat-inact, GDNF, 200 ng/ml, white circles) (n = 8), induced an increase in the firing frequency of neurons. The horizontal bar depicts the application duration of GDNF or its inactivated form. For inactivation, GDNF was heated at 95°C for 1 hr. D, Dose-response of GDNF-induced enhancement of firing rate. * p < 0.05 vs. baseline. n = 10, 8, 15 for 50, 100, and 200 ng/ml GDNF, respectively. E–F, GDNF enhancement of the spontaneous firing rate of VTA neurons requires the activation of the MAPK pathway, but not the PI-3K pathway. Slices were pretreated with PD 98059 (10 μM), or LY 294002 (25 μM), for 45 min and GDNF’s (200 ng/ml) effect on the firing rate was tested in the continuous presence of the inhibitors. E, Firing rate of neurons in the presence of PD 98059 before and during GDNF application. F, Bar graph summarizing the effect of PD 98059 and LY 294002 on the firing rate of neurons in response to GDNF. * p < 0.05 vs. baseline. n = 9 (PD 98059), n = 11 (LY 294002). p = 0.15 for the difference in firing rate before and during GDNF application in the continuous presence of PD 98059. See also Figure S3.

Article Snippet: Recombinant human GDNF was obtained from R&D System (Minneapolis, MN).

Techniques: Construct, Activation Assay

Journal:

Article Title: Nucleus Accumbens-Derived GDNF is a Retrograde Enhancer of Dopaminergic Tone in the Mesocorticolimbic System

doi: 10.1523/JNEUROSCI.3909-10.2010

Figure Lengend Snippet: A–B, GDNF (10 μg/ 2μl) was bilaterally infused into the NAc 7-11 days following intra- PFC infusions of DiI. VTA slices were prepared 12 hrs after GDNF infusion, and the spontaneous firing of DiI-labled PFC-projecting neurons was measured. A, A representative coronal section confirming the injection sites within the PFC. Arrowheads indicate DiI deposit. Scale bar, 0.5 mm B, Cumulative probability plot comparing spontaneous firing rates of individual neurons in slices from vehicle- (black circles) and GDNF- (red circles) treated rats. ** p < 0.01 vs. vehicle by Kolmogorov-Smirnov test, n = 22 cells from 5 rats for each group.

Article Snippet: Recombinant human GDNF was obtained from R&D System (Minneapolis, MN).

Techniques: Injection

Journal:

Article Title: Nucleus Accumbens-Derived GDNF is a Retrograde Enhancer of Dopaminergic Tone in the Mesocorticolimbic System

doi: 10.1523/JNEUROSCI.3909-10.2010

Figure Lengend Snippet: 6-OHDA was unilaterally (A–B) or bilaterally (C) infused into the NAc. For electrophysiology experiments (C), DiI was also bilaterally infused into the PFC. Three weeks after the infusion of 6-OHDA, vehicle (A) or GDNF (10 μg/2 μl) (B,C) was bilaterally infused into the NAc. Twelve hrs after GDNF infusion, animals were perfused and brains removed to examine the immunoreactivity of TH (A) and phosphorylated ERK1/2 (p-ERK1/2) (B). The spontaneous firing of DiI-labeled PFC-projecting VTA neurons was measured in parallel (C). A, Image depicts TH staining in the whole striatum after 6-OHDA (left) and vehicle (right) infusion into the NAc. The reduction of TH level in the area defined by the dash line, contains the most part of the NAc. Scale bar, 1 mm. B, Intra-NAc infusion of 6-OHDA reduces subsequent GDNF-mediated increase in p-ERK1/2 levels in the VTA. Image shows ERK1/2 phosphorylation in the VTA after unilateral 6-OHDA (left) and subsequent bilateral GDNF infusions into the NAc. Scale bar, 500 μm. C, 6-OHDA lesion of DAergic fibers in the NAc abolishes NAc-derived GDNF enhancement of the spontaneous firing rate of PFC-projecting VTA neurons. Cumulative probability plot comparing the firing rate of neurons from GDNF (red) and vehicle (black)-infused animals. n = 39 (vehicle) and 42 (GDNF). p > 0.05 Kolmogorov-Smirnov test.

Article Snippet: Recombinant human GDNF was obtained from R&D System (Minneapolis, MN).

Techniques: Labeling, Staining, Phospho-proteomics, Derivative Assay

Journal:

Article Title: Nucleus Accumbens-Derived GDNF is a Retrograde Enhancer of Dopaminergic Tone in the Mesocorticolimbic System

doi: 10.1523/JNEUROSCI.3909-10.2010

Figure Lengend Snippet: A VTA DA neuron (yellow) innervates a medium spiny neuron (MSN, Green), the principal cell of the NAc. A neighboring VTA DA neuron (orange) projects to the PFC (grey). GDNF (red triangle) is synthesized in and released by the MSN (A). The polypeptide is taken up by the terminal of the NAc-projecting VTA DA neuron (B). GDNF is then retrogradely transported into the neuronal soma and/or dendrite located in the VTA, where GDNF is secreted (C). The released GDNF binds to GFRα-1 (white) localized on the membrane of the same and/or adjacent NAc-projecting (D) and PFC-projecting (E) VTA neuron, which leads to the ligation of the GDNF/GFRα-1 complex to Ret (green), leading to its activation (D and E). Activation of Ret results in the activation of ERK1/2 (blue, F), that in turn causes increased spontaneous neuronal firing (G and H), which propagates along the axon back to the DA terminal in the NAc, leading to the elevation of DA (black) release (I).

Article Snippet: Recombinant human GDNF was obtained from R&D System (Minneapolis, MN).

Techniques: Synthesized, Membrane, Ligation, Activation 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: 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: JNCI Journal of the National Cancer Institute

Article Title: Paracrine Regulation of Pancreatic Cancer Cell Invasion by Peripheral Nerves

doi: 10.1093/jnci/djp456

Figure Lengend Snippet: Glial cell-derived neurotrophic factor–induced cancer cell invasion. A) Serum-starved MiaPaCa2 cells were plated in the upper chamber, and GDNF (1–100 ng/mL) was added to the lower chamber of transwell plates and cultured for 24 hours (white bars). In another experiment, dorsal root ganglion (DRG) nerve cells were grown on the lower plate instead of adding GDNF (the condition is indicated below the graph). While nerves induced cancer cell migration (control—blue bar), adding anti-GDNF antibodies to the lower chamber suppressed it (red bars). Nonimmune Igs were used for control (n = 10). Yellow bars: lentiviral gene transduction of neurons with short hairpin RNA (shRNA) targeting GDNF expression (shGDNF) also inhibited cancer cell migration toward the neurons (n = 6). An empty viral vector served as the control (shControl). B) Immunoblots of conditioned media from dissociated DRG nerve cell cultures. Conditioned media was recovered at 24–48 hours after incubation. Protein analysis showed detection of a protein band, which corresponds to the secreted form of GDNF. C) Immunoblots of GDNF protein recovered from nerve cell lysates after lentiviral gene transduction with shRNA directed against GDNF (n = 3–6 experiments in each condition). Empty vector lentivirus was used as control (shControl). D) Cancer cells (green asterisk) were grown in Matrigel adjacent to DRG (white asterisk). Pictures show representative experiments of neural invasion by MiaPaCa2 cells taken at day 10 in control conditions allowing for neural invasion (left); with anti-GDNF antibodies inhibiting invasion (middle) and of a DRG from a heterozygous mouse deficient of GDNF (gdnf−/+). The black arrows indicate the invading cancer cells. E) An illustration showing the calculation of nerve invasion index (α/β). F) Dose–response curves showing the effect of anti-GDNF antibodies on invasion index at day 10 (P < .001; n = 5–15 experiments in each condition). Nonimmune Igs served as control (open circle). G) Nerve invasion index in wild-type (WT; white bar) and gdnf−/+ mice (gray bar) at day 10 (n = 60–66). P values in (A, F, and G) were calculated by two-sided Student t test. DMEM = Dulbecco's modified Eagle medium; hpf = high-power field.

Article Snippet: Lentiviral Gene Transduction and RNA Interference for Inhibition of GDNF Expression by DRG Lentiviral pLKO.1 vectors expressing short hairpin RNA against murine GDNF ( {"type":"entrez-nucleotide","attrs":{"text":"NM_010275","term_id":"672349279"}} NM_010275 ) were purchased from Open Biosystems (Huntsville, AL) and from OriGene Technologies (Rockville, MD). pLKO.1 empty vector was used as a negative control.

Techniques: Derivative Assay, Cell Culture, Migration, Transduction, shRNA, Expressing, Plasmid Preparation, Western Blot, Incubation, Modification

Journal: JNCI Journal of the National Cancer Institute

Article Title: Paracrine Regulation of Pancreatic Cancer Cell Invasion by Peripheral Nerves

doi: 10.1093/jnci/djp456

Figure Lengend Snippet: Activation of RET and mitogen-activated protein kinase in neuroinvasive cancer cells. A) Expression of RET (170/150 kDa) and GFRα1 (53 kDa) in various cancer cell lines, as determined by immunoblotting. Expression of β-actin (42 kDa) was used as a control for loading and transfer. B) Cancer cell colonies (green asterisk) were grown in Matrigel adjacent to dorsal root ganglion (DRG) (white asterisk). When the DRG neurites made contact with the cancer colony, anti-RET antibodies (1 μg/mL) or recombinant mouse RET-Fc chimera (5 μg/mL) were added to the media. Pictures show representative experiments of neural invasion by MiaPaCa2 cells taken at day 10. Nonimmune antibodies were used as control (n = 8–12). C) The effect of small interfering RNA (siRNA) directed against RET (siRET) on invasion of pancreatic cancer cells along DRG nerves. siRET reduced RET expression within 72 hours after transfection (immunoblotting—lower panel), whereas noncoding siRNA served as control (siControl) and had no impact on RET expression. Nerve invasion was measured in vitro on day 7 (n = 11–17). D) Dose–response effect of the RET inhibitor pyrazolopyrimidine-1 (PYP1) on MiaPaCa2 cell invasion index. Inset: effect of PYP1 on cell migration toward DRG nerve cells in dual chamber migration assays (high-power field [hpf], n = 10). E) Dose–response curves of the effect of the MEK-1 inhibitor PD98059 on MiaPaCa2 nerve invasion index (P < .001; n = 5–15 experiments in each condition). F) Effect of PYP1 and PD98059 on invasion index of another human pancreatic adenocarcinoma cell line—Panc1, and on a human cervical cancer cell line—HeLa (*P < .001; n = 10 experiments in each condition). G) Cell viability was measured after treatment with PD98059 (25 μM) and PYP1 (2 μM) (n = 3 in each condition). H) Immunoblotting to assay GDNF expression by the DRG after treatment with PD98059 and PYP1. P values were calculated by using a two-sided Student t test.

Article Snippet: Lentiviral Gene Transduction and RNA Interference for Inhibition of GDNF Expression by DRG Lentiviral pLKO.1 vectors expressing short hairpin RNA against murine GDNF ( {"type":"entrez-nucleotide","attrs":{"text":"NM_010275","term_id":"672349279"}} NM_010275 ) were purchased from Open Biosystems (Huntsville, AL) and from OriGene Technologies (Rockville, MD). pLKO.1 empty vector was used as a negative control.

Techniques: Activation Assay, Expressing, Western Blot, Recombinant, Small Interfering RNA, Transfection, In Vitro, Migration