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: EBioMedicine

Article Title: Unmasking a new prognostic marker and therapeutic target from the GDNF-RET/PIT1/p14ARF/p53 pathway in acromegaly

doi: 10.1016/j.ebiom.2019.04.007

Figure Lengend Snippet: Primary cultures of human acromegaly in humanized conditions (h7H) unveiled Sorafenib as a potential new treatment. a) Acromegaly (P-ACRO28, P-ACRO30 and P-ACRO32) and NFPA (P-NFPA41) cultured in h7H conditions grew for many passages while maintaining mRNA expression of key genes from : in ACRO, GH, PIT1, GHRHR, RETL and RETS isoforms, SSTR2 and 5; in NFPA, SF1. Data are normalized to the first acromegaly P-ACRO28. Passage was performed by splitting the culture in two (shown by small ‘p’). b) Left: Human GH (hGH) was secreted by cultured ACRO into the medium as demonstrated by western blot. Centre: Quantitative hGH (pg/mL) measurements performed in whole medium taken just before passaging. Right: Secretion was normalized by cell count and length of incubation with the cells (3–4 days). c) Human GDNF (hGDNF) was secreted into the medium by ACRO, but not by NFPA, as demonstrated by ELISA. Secretion was increased as cells grew, as shown for ACRO32. d) When cells were deprived of GDNF, RET processing induced apoptosis (white bar) that was blocked by addition of GDNF (hatched white bars). Five TKIs used for other neuroendocrine tumors (Vandetanib (V), Lenvatinib (L), Sunitinib (Su), Cabozantinib (C) and Sorafenib (So)) were tested against the survival action of GDNF at clinically relevant concentrations. Three independent cultures are shown: P-ACRO28, P-ACRO30 and P-ACRO32. Some of the inhibitors enhanced RET-dependent apoptosis in the absence of GDNF (black bars) but did not have a strong effect on GDNF-induced survival (black hatched bars). Sorafenib was the only TKI that potently blocked the GDNF survival effect in the three ACRO without exacerbating RET apoptosis in the absence of GDNF (which we assumed to be a toxic effect). e) Dose-response curve of Sorafenib in P-ACRO30 and P-ACRO32, demonstrating a GDNF-counteracting effect at lower doses than those used in cancer treatment. f) Sorafenib could also block the survival effect of NRTN and combined GDNF+NRTN. g) Sorafenib could block the survival effect of GDNF on both human RETL and RETS isoforms, transfected in the non-RET-expressing rat pituitary somatotroph cell line GH4C1. (n.d. = not detected). (d-g: Mean ± SEM. ANOVA test, all bars compared to white bar –deprived in the absence of GDNF-) (*, p < 0·05; **, p < 0·01; ***, p < 0·001; ****, p < 0·0001).

Article Snippet: Secreted GDNF levels were measured with a Human GDNF DuoSet ELISA (DY212, R&D Systems, USA) according to the manufacturer's protocol.

Techniques: Cell Culture, Expressing, Western Blot, Passaging, Cell Counting, Incubation, Enzyme-linked Immunosorbent Assay, Blocking Assay, Transfection

Journal: EBioMedicine

Article Title: Unmasking a new prognostic marker and therapeutic target from the GDNF-RET/PIT1/p14ARF/p53 pathway in acromegaly

doi: 10.1016/j.ebiom.2019.04.007

Figure Lengend Snippet: mRNA expression characterizing ACRO and the RET pathway. a) Quantitative RNA expression comparing mean ± SEM ACRO ( n = 32, blue bars) and NFPA ( n = 56–63, pink bars). In every sample, gene expression was normalized to a commercial pool of pituitary poly-A mRNA (technical control; white bars). Genes most highly expressed in ACRO were characteristic of functional pituitary somatotrophs, related to GH secretion (GH Taq (22 KDa), GH Sybr (22, 20 and 17 KDa)), or PIT1 and hypothalamic regulation (GHRHR, SSTR2, SSTR5). However, the RET receptor (RETN), its ligand GDNF and genes in the RET pathway regulated at the RNA level (ARF and PIT1) were significantly more abundant in ACRO than NFPA. p53 was slightly upregulated in ACRO. SF-1 was characteristic of NFPA, and T-PIT of ACTH-secreting adenomas (orange bar). (Mann-Whitney test). b) Cartoon representing isoforms of the RET receptor, differing at the C-terminal tail (long RETL and short RETS), its four ligands and its GFR-alpha co-receptors (GFRA1–4). Although there is preference of a ligand for a co-receptor, cross-interaction exists. In somatotrophs in the absence of ligand, RET is processed by Caspase-3 generating an intracellular fragment (IC-RET) that triggers a cell-death pathway through overexpression of PIT1 gene inducing p14ARF expression, p53 accumulation and apoptosis. When the ligand is present, RET dimerizes and activates its cytoplasmic tyrosine kinase activity leading to AKT phosphorylation and survival. c) In ACRO, both RETL and RETS are expressed in approximately equal amounts. Although all four RET co-receptors were expressed, the most highly expressed was GFRA1 (high affinity for GDNF), followed by GFRA4. (ANOVA). d) GDNF was by far the most highly expressed ligand, with NTRN and PSPN also abundantly expressed. (ANOVA). e) Significant correlations among all the genes studied in ACRO (r s > 0·36) revealed that GDNF expression was significantly and positively correlated with expression of RET isoforms and the other ligands. GDNF (and NRTN) were negatively correlated with PIT1 expression but positively correlated with PROP1. As expected, expression of both transcription factors PIT1 and PROP1 were negatively correlated. PROP1 was also correlated with GFRA1 and SF1. ARF expression was significantly correlated with the somatotroph phenotype (GH, GHRHR, AIP), T-PIT and the ligand ARTN. (Spearman test). Weakest correlations were lost ( p > .2) when the naïve group ( N = 16) was analysed separately from the group receiving pre-surgery therapy (N = 16). Yellow dots: correlations lost in pre-surgery therapy group; Blue dots: correlations lost in naïve group. f) Genes implicated in lost correlations were not differentially expressed between both groups except for PIT1 and GH that were significantly reduced in the pre-surgery therapy group (all Mann-Whitney test except ARF t -Test). ( p < 0·05; **, p < 0·01; ***, p < 0·001; ****, p < 0·0001; 0·0000 means lower p). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Secreted GDNF levels were measured with a Human GDNF DuoSet ELISA (DY212, R&D Systems, USA) according to the manufacturer's protocol.

Techniques: Expressing, RNA Expression, Gene Expression, Control, Functional Assay, MANN-WHITNEY, Over Expression, Activity Assay, Phospho-proteomics

Journal: EBioMedicine

Article Title: Unmasking a new prognostic marker and therapeutic target from the GDNF-RET/PIT1/p14ARF/p53 pathway in acromegaly

doi: 10.1016/j.ebiom.2019.04.007

Figure Lengend Snippet: ARF mRNA expression as a prognostic marker for ACRO resistant to first-line combined treatment (sugery + fgSSA). GFRA4 as a prognostic marker only for those not cured by surgery. a-b: Significant correlations (Spearman test, rs > 0·5) between mRNA expression and clinical variables (diagnostic, pathologic and prognostic/follow-up) (See also Supplementary Table 1). Dots: blue, correlations not existing in naïve samples; yellow: correlations lost in the pre-surgery therapy group. a) Left: serum GH at diagnosis was correlated with GH, SSTR2 (only if pre-treated), AIP and p53. Centre: Pre-surgery therapy was negatively correlated with GH and PIT1. ARF was negatively correlated with the negative characteristics of the tumor (invasiveness (only if naïve), Knosp grade, residual tumor). Right: TPIT was negatively correlated with negative tumor characteristics (diameter, volume (only if pre-treated), invasiveness and Knosp) but positively correlated with surgical cure. b) Left: ARF was the strongest positive marker for response to adjuvant treatment with fgSSA (rs > 0·8). GFRA4 was the second-strongest marker but negatively correlated with response (rs > 0·7). GH, TPIT and SSTR2 (only if naïve) showed some positive correlation with response to analogs (rs 0·5–0·6). Right: Combining first-line treatments (surgery+fgSSA) into a single category, ARF (rs > 0·7) and GFRA4 (rs = 0·5) were the most significant positively and negatively correlated markers, respectively, followed by TPIT (rs = 0·6) as a positive marker. c-f: Patients were categorized into two groups, Group 0 (Resistant: Not cured by surgery and resistant to analogs) and Group 1 (Responsive to first-line treatment, either cured by surgery or controlled with analogs). A subanalysis of patients not cured by surgery was also performed. c) ARF expression was a good discriminator of Group 0 and Group 1, both in the series as a whole ( p < 0·0001) and in patients not cured by surgery ( p = 0·0002). d) Samples were categorized with different cutoffs for non-mutated (GNASwt cutoff 0·1) or mutated (GNASmut, cutoff 0·06) GNAS; Chi-squared tests classified all samples. e) A subanalysis of naïve patients and those with pre-surgery therapy including categorizing by GNAS demonstrated the ability of ARF to separate Group 0 from Group 1 in either group of patients with similar cutoffs. f) Left: Enhanced GFRA4 expression was not a good classifier for ACRO as some in Group 1 (Responsive, high ARF) had high GFRA4 expression. Right: In the group of non-surgically cured, GFRA4 was a good classifier in the opposite way to ARF, with high levels in Group 0 (Resistant to first-line therapy). Coloured dots show reoperations after radiotherapy (Group 0, yellow; Group 1, orange). g) Western blots of RET pathway (RET, PIT1, ARF, p53) and ligand (GDNF, NRTN) proteins with controls (GH, GAPDH, ACTB) from ACRO tissue extracts (Group 1 (Responsive) and Group 0 (Resistant)). ARF and GFRa4 protein levels corroborated mRNA levels described above. ACRO27 (Group 0) expressed high RET but low PIT-1 protein levels, correlating with absence of p14ARF and p53. GFRA4 showed three different molecular weights corresponding to distinct isoforms. The RET co-receptor (GPI, canonical isoform) was highly expressed in ACRO27 (Group 0) while ACRO7 and ACRO8 (Group 1) expressed different non-RET co-receptor isoforms. Quantification of ARF protein band intensity respect to controls, ACTB (yellow bars) or GAPDH (orange bars), in relation to the ARF mRNA expression (blue bars). (c-e-f Mann-Whitney test; d Chi-square test. *, p < 0·05; **, p < 0·01; ***, p < 0·001; ****, p < 0·0001; 0·0000 means lower p). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Secreted GDNF levels were measured with a Human GDNF DuoSet ELISA (DY212, R&D Systems, USA) according to the manufacturer's protocol.

Techniques: Expressing, Marker, Diagnostic Assay, Biomarker Discovery, Adjuvant, Western Blot, MANN-WHITNEY

Journal: EBioMedicine

Article Title: Unmasking a new prognostic marker and therapeutic target from the GDNF-RET/PIT1/p14ARF/p53 pathway in acromegaly

doi: 10.1016/j.ebiom.2019.04.007

Figure Lengend Snippet: Blocking of RET signal transduction by Sorafenib exchanged the RET-GDNF survival pathway for the RET apoptotic pathway. a) Antibody array performed with extracts from P-ACRO30 and P-ACRO32 following one- hour incubations in each of four conditions: deprivation (no GDNF), GDNF (500 ng/mL), Sorafenib (4 microg/mL) or Sorafenib+GDNF. b) Signal quantification (Mean ± SEM. ANOVA test): GDNF induced AKT/MTOR phosphorylation, and downregulated active p-p53 (Ser15 p-p53), as well as cleaved PARP (cl-PARP) and Caspase-3 (cl-CASP3), hallmarks of apoptosis. Sorafenib blocked the AKT pathway, inducing phosphorylation of AMPK, p53 and apoptotic markers. c-e) Western blots with different cell extracts confirmed the results of the array. c) At 1 h in membrane extracts, GDNF induced full-length RET phosphorylation and blocked Caspase-3 cleavage; both were prevented by Sorafenib. d) At 1 h in cytoplasmic extracts, GDNF phosphorylated AKT, mTOR and, less intensely, S6K while preventing p53 phosphorylation. In the presence of Sorafenib, GDNF was unable to activate the AKT/MTOR pathway, but induced phosphorylation of AMPK and p53. As expected, the caspase-processed cytoplasmic fragment of RET (IC-RET) was strongly expressed in deprived cells in the absence of GDNF, reduced in the presence of GDNF and recovered with Sorafenib. e) In cells deprived of GDNF for 24 h, PIT1 was induced, activating ARF expression and p53 accumulation, thus leading to apoptosis. GDNF reduced PIT1, ARF and p53 expression, all of which recovered in the presence of Sorafenib. (*, p < 0·05; **, p < 0·01; ***, p < 0·001).

Article Snippet: Secreted GDNF levels were measured with a Human GDNF DuoSet ELISA (DY212, R&D Systems, USA) according to the manufacturer's protocol.

Techniques: Blocking Assay, Transduction, Ab Array, Phospho-proteomics, Western Blot, Membrane, Expressing