Effect of GDNF on neuroblast proliferation and photoreceptor survival: additive protection with docosahexaenoic acid

Purpose. In a previous study, it was reported that docosahexaenoic acid (DHA) is essential to postpone apoptosis and to promote differentiation of rat retina photoreceptors in vitro. In the current study, the protective effects of GDNF on photoreceptor cells during development in vitro and its action when combined with DHA were investigated. Methods. Rat retina neuronal cultures were incubated in a chemically defined medium, either without photoreceptor survival factors or supplemented with GDNF, DHA, or GDNF plus DHA. Evolution of survival, apoptosis, opsin expression, mitochondrial functioning, and cell proliferation were investigated at different times of development in vitro. Results. Incubation with GDNF selectively increased the number of surviving photoreceptors, reduced their apoptosis, and augmented opsin expression. Proliferative cell nuclei antigen (PCNA) determination and addition of [3H]-thymidine or bromodeoxyuridine showed that GDNF promoted neuroblast proliferation during the first hours of development in vitro. The combined addition of GDNF and DHA enhanced opsin expression and photoreceptor survival in an additive manner. The advance of photoreceptor apoptosis in cultures without trophic factors correlated with an increased impairment in mitochondrial functionality. Addition of GDNF and DHA significantly diminished the loss of mitochondrial activity. Conclusions. These results show that GDNF stimulated the cell cycle progression, leading to neuroblast proliferation at early stages of development, and delayed the onset of apoptosis later on, improving differentiation and acting as a trophic factor for photoreceptors. The combination of GDNF with DHA had an additive effect both on photoreceptor survival and on opsin expression. Preservation of mitochondrial function may be involved in the antiapoptotic effect of both factors.Instituto Multidisciplinario de Biología Celula

Effect of GDNF on neuroblast proliferation and photoreceptor survival: additive protection with docosahexaenoic acid

Purpose. In a previous study, it was reported that docosahexaenoic acid (DHA) is essential to postpone apoptosis and to promote differentiation of rat retina photoreceptors in vitro. In the current study, the protective effects of GDNF on photoreceptor cells during development in vitro and its action when combined with DHA were investigated. Methods. Rat retina neuronal cultures were incubated in a chemically defined medium, either without photoreceptor survival factors or supplemented with GDNF, DHA, or GDNF plus DHA. Evolution of survival, apoptosis, opsin expression, mitochondrial functioning, and cell proliferation were investigated at different times of development in vitro. Results. Incubation with GDNF selectively increased the number of surviving photoreceptors, reduced their apoptosis, and augmented opsin expression. Proliferative cell nuclei antigen (PCNA) determination and addition of [3H]-thymidine or bromodeoxyuridine showed that GDNF promoted neuroblast proliferation during the first hours of development in vitro. The combined addition of GDNF and DHA enhanced opsin expression and photoreceptor survival in an additive manner. The advance of photoreceptor apoptosis in cultures without trophic factors correlated with an increased impairment in mitochondrial functionality. Addition of GDNF and DHA significantly diminished the loss of mitochondrial activity. Conclusions. These results show that GDNF stimulated the cell cycle progression, leading to neuroblast proliferation at early stages of development, and delayed the onset of apoptosis later on, improving differentiation and acting as a trophic factor for photoreceptors. The combination of GDNF with DHA had an additive effect both on photoreceptor survival and on opsin expression. Preservation of mitochondrial function may be involved in the antiapoptotic effect of both factors.Instituto Multidisciplinario de Biología Celula

Effect of GDNF on neuroblast proliferation and photoreceptor survival: additive protection with docosahexaenoic acid

Purpose. In a previous study, it was reported that docosahexaenoic acid (DHA) is essential to postpone apoptosis and to promote differentiation of rat retina photoreceptors in vitro. In the current study, the protective effects of GDNF on photoreceptor cells during development in vitro and its action when combined with DHA were investigated. Methods. Rat retina neuronal cultures were incubated in a chemically defined medium, either without photoreceptor survival factors or supplemented with GDNF, DHA, or GDNF plus DHA. Evolution of survival, apoptosis, opsin expression, mitochondrial functioning, and cell proliferation were investigated at different times of development in vitro. Results. Incubation with GDNF selectively increased the number of surviving photoreceptors, reduced their apoptosis, and augmented opsin expression. Proliferative cell nuclei antigen (PCNA) determination and addition of [3H]-thymidine or bromodeoxyuridine showed that GDNF promoted neuroblast proliferation during the first hours of development in vitro. The combined addition of GDNF and DHA enhanced opsin expression and photoreceptor survival in an additive manner. The advance of photoreceptor apoptosis in cultures without trophic factors correlated with an increased impairment in mitochondrial functionality. Addition of GDNF and DHA significantly diminished the loss of mitochondrial activity. Conclusions. These results show that GDNF stimulated the cell cycle progression, leading to neuroblast proliferation at early stages of development, and delayed the onset of apoptosis later on, improving differentiation and acting as a trophic factor for photoreceptors. The combination of GDNF with DHA had an additive effect both on photoreceptor survival and on opsin expression. Preservation of mitochondrial function may be involved in the antiapoptotic effect of both factors.Instituto Multidisciplinario de Biología Celula

In vitro and in vivo herpetic vector-mediated gene transfer in the pituitary gland: Impact on hormone secretion

Objective: Herpes simplex virus type 1 (HSV-1)-derived vectors are known to be effective tools to deliver transgenes into normal and neoplastic anterior pituitary (AP) cells in vitro. Our objective was to assess the in vitro and in vivo effects of tsK/β-gal, a temperature-sensitive HSV-1-derived vector harbouring the E. coli β-galactosidase gene, on AP hormone secretion as well as on transgene expression in rat AP tumours (hyperplastic prolactinomas). Design: The impact of vector infection on prolactin (PRL) and GH release was determined in vitro in normal and hyperplastic (lactotrophic) dispersed AP cells exposed for 24 h to tsK/β-gal as well as in vivo in ectopic AP grafts. In some oestrogen-induced prolactinoma-carrying rats, vector suspension was stereotaxically injected into the glands to assess transgene expression in vivo. Methods: GH and PRL release was measured by specific RIAs. In vivo transgene expression was assessed by immunohistochemistry for β-galactosidase and enzymohistochemistry (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). Ectopic pituitary grafts and stereotaxic surgery were performed following standard procedures. Results: At a multiplicity of infection of 0.5, the vector induced a 30 and 22% fall in PRL and GH release respectively in normal AP cells, whereas the corresponding hormone release inhibition for hyperplastic AP cells was 41 and 33% for PRL and GH respectively. In ectopic pituitary grafts, the effect of vector infection on hormone secretion was assessed by measuring serum PRL levels in the host rats every 5 days for 4 weeks post-grafting. In the pituitary-grafted rats that received the viral vector, serum PRL failed to increase to the levels achieved in control-grafted animals. Finally, pituitary tumours stereotaxically injected with tsK/β-gal showed widespread expression of the β-galactosidase transgene around the injection areas. Conclusions: The results reported here have implications for basic studies using gene transfer to pituitary gland as well as potential gene therapy approaches to pituitary diseases.Instituto de Investigaciones Bioquímicas de La PlataInstituto de Biotecnologia y Biologia MolecularInstituto Multidisciplinario de Biología Celula

In vitro and in vivo herpetic vector-mediated gene transfer in the pituitary gland: Impact on hormone secretion

Objective: Herpes simplex virus type 1 (HSV-1)-derived vectors are known to be effective tools to deliver transgenes into normal and neoplastic anterior pituitary (AP) cells in vitro. Our objective was to assess the in vitro and in vivo effects of tsK/β-gal, a temperature-sensitive HSV-1-derived vector harbouring the E. coli β-galactosidase gene, on AP hormone secretion as well as on transgene expression in rat AP tumours (hyperplastic prolactinomas). Design: The impact of vector infection on prolactin (PRL) and GH release was determined in vitro in normal and hyperplastic (lactotrophic) dispersed AP cells exposed for 24 h to tsK/β-gal as well as in vivo in ectopic AP grafts. In some oestrogen-induced prolactinoma-carrying rats, vector suspension was stereotaxically injected into the glands to assess transgene expression in vivo. Methods: GH and PRL release was measured by specific RIAs. In vivo transgene expression was assessed by immunohistochemistry for β-galactosidase and enzymohistochemistry (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). Ectopic pituitary grafts and stereotaxic surgery were performed following standard procedures. Results: At a multiplicity of infection of 0.5, the vector induced a 30 and 22% fall in PRL and GH release respectively in normal AP cells, whereas the corresponding hormone release inhibition for hyperplastic AP cells was 41 and 33% for PRL and GH respectively. In ectopic pituitary grafts, the effect of vector infection on hormone secretion was assessed by measuring serum PRL levels in the host rats every 5 days for 4 weeks post-grafting. In the pituitary-grafted rats that received the viral vector, serum PRL failed to increase to the levels achieved in control-grafted animals. Finally, pituitary tumours stereotaxically injected with tsK/β-gal showed widespread expression of the β-galactosidase transgene around the injection areas. Conclusions: The results reported here have implications for basic studies using gene transfer to pituitary gland as well as potential gene therapy approaches to pituitary diseases.Instituto de Investigaciones Bioquímicas de La PlataInstituto de Biotecnologia y Biologia MolecularInstituto Multidisciplinario de Biología Celula

In vitro and in vivo herpetic vector-mediated gene transfer in the pituitary gland: Impact on hormone secretion

Objective: Herpes simplex virus type 1 (HSV-1)-derived vectors are known to be effective tools to deliver transgenes into normal and neoplastic anterior pituitary (AP) cells in vitro. Our objective was to assess the in vitro and in vivo effects of tsK/β-gal, a temperature-sensitive HSV-1-derived vector harbouring the E. coli β-galactosidase gene, on AP hormone secretion as well as on transgene expression in rat AP tumours (hyperplastic prolactinomas). Design: The impact of vector infection on prolactin (PRL) and GH release was determined in vitro in normal and hyperplastic (lactotrophic) dispersed AP cells exposed for 24 h to tsK/β-gal as well as in vivo in ectopic AP grafts. In some oestrogen-induced prolactinoma-carrying rats, vector suspension was stereotaxically injected into the glands to assess transgene expression in vivo. Methods: GH and PRL release was measured by specific RIAs. In vivo transgene expression was assessed by immunohistochemistry for β-galactosidase and enzymohistochemistry (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). Ectopic pituitary grafts and stereotaxic surgery were performed following standard procedures. Results: At a multiplicity of infection of 0.5, the vector induced a 30 and 22% fall in PRL and GH release respectively in normal AP cells, whereas the corresponding hormone release inhibition for hyperplastic AP cells was 41 and 33% for PRL and GH respectively. In ectopic pituitary grafts, the effect of vector infection on hormone secretion was assessed by measuring serum PRL levels in the host rats every 5 days for 4 weeks post-grafting. In the pituitary-grafted rats that received the viral vector, serum PRL failed to increase to the levels achieved in control-grafted animals. Finally, pituitary tumours stereotaxically injected with tsK/β-gal showed widespread expression of the β-galactosidase transgene around the injection areas. Conclusions: The results reported here have implications for basic studies using gene transfer to pituitary gland as well as potential gene therapy approaches to pituitary diseases.Instituto de Investigaciones Bioquímicas de La PlataInstituto de Biotecnologia y Biologia MolecularInstituto Multidisciplinario de Biología Celula

EphA3 Expressed in the Chicken Tectum Stimulates Nasal Retinal Ganglion Cell Axon Growth and Is Required for Retinotectal Topographic Map Formation

BACKGROUND: Retinotopic projection onto the tectum/colliculus constitutes the most studied model of topographic mapping and Eph receptors and their ligands, the ephrins, are the best characterized molecular system involved in this process. Ephrin-As, expressed in an increasing rostro-caudal gradient in the tectum/colliculus, repel temporal retinal ganglion cell (RGC) axons from the caudal tectum and inhibit their branching posterior to their termination zones. However, there are conflicting data regarding the nature of the second force that guides nasal axons to invade and branch only in the caudal tectum/colliculus. The predominant model postulates that this second force is produced by a decreasing rostro-caudal gradient of EphA7 which repels nasal optic fibers and prevents their branching in the rostral tectum/colliculus. However, as optic fibers invade the tectum/colliculus growing throughout this gradient, this model cannot explain how the axons grow throughout this repellent molecule. METHODOLOGY/PRINCIPAL FINDINGS: By using chicken retinal cultures we showed that EphA3 ectodomain stimulates nasal RGC axon growth in a concentration dependent way. Moreover, we showed that nasal axons choose growing on EphA3-expressing cells and that EphA3 diminishes the density of interstitial filopodia in nasal RGC axons. Accordingly, in vivo EphA3 ectodomain misexpression directs nasal optic fibers toward the caudal tectum preventing their branching in the rostral tectum. CONCLUSIONS: We demonstrated in vitro and in vivo that EphA3 ectodomain (which is expressed in a decreasing rostro-caudal gradient in the tectum) is necessary for topographic mapping by stimulating the nasal axon growth toward the caudal tectum and inhibiting their branching in the rostral tectum. Furthermore, the ability of EphA3 of stimulating axon growth allows understanding how optic fibers invade the tectum growing throughout this molecular gradient. Therefore, opposing tectal gradients of repellent ephrin-As and of axon growth stimulating EphA3 complement each other to map optic fibers along the rostro-caudal tectal axis

Glial cell line-derived neurotrophic factor gene therapy ameliorates chronic hyperprolactinemia in senile rats

Progressive dysfunction of hypothalamic tu-beroinfundibular dopaminergic (TIDA) neurons during nor-mal aging is associated in the female rat with chronic hyper-prolactinemia. We assessed the effectiveness of glial cellline-derived neurotrophic factor (GDNF) gene therapy to re-store TIDA neuron function in senile female rats and reversetheir chronic hyperprolactinemia. Young (2.5 months) andsenile (29 months) rats received a bilateral intrahypothalamicinjection (1010pfu) of either an adenoviral vector expressingthe gene for β-galactosidase; (Y-βgal and S-βgal, respectively) or a vector expressing rat GDNF (Y-GDNF and S-GDNF,respectively). Transgenic GDNF levels in supernatants ofGDNF adenovector-transduced N2a neuronal cell cultureswere 25±4 ng/ml, as determined by bioassay. In the rats,serum prolactin (PRL) was measured at regular intervals. Onday 17 animals were sacrificed and neuronal nuclear antigen(NeuN) and tyrosine hydroxylase (TH) immunoreactive cellscounted in the arcuate–periventricular hypothalamic region.The S-GDNF but not the S-βgal rats, showed a significantreduction in body weight. The chronic hyperprolactinemia ofthe senile females was significantly ameliorated in the S-GDNF rats (P<0.05) but not in the S-βgal rats. Neither age norGDNF induced significant changes in the number of NeuNand TH neurons. We conclude that transgenic GDNF amelio-rates chronic hyperprolactinemia in aging female rats, prob-ably by restoring TIDA neuron function.Instituto Multidisciplinario de Biología CelularInstituto de Investigaciones Bioquímicas de La Plat

EphA3 ectodomain stimulates nasal RGC axon growth in vitro.

<p>(A, B) Microphotographs of nasal retinal explants grown on clustered Fc (A) or clustered EphA3-Fc at 2 nM (B). RGC axons grow longer on EphA3-Fc. Scale bars  = 20 µm. (C–D) Quantification of axon length of nasal (C) and temporal explants (D) grown on substrates formed by laminin and clustered Fc or EphA3-Fc at different concentrations. Axon length is indicated in µm and concentrations are indicated in nM of Fc or EphA3-Fc. Temporal explants grow longer axons than nasal ones in control conditions (p: 0.0002, compare first bar of nasal RGCs in C with first bar of temporal RGCs in D). EphA3-Fc increased nasal RGCs axon growth from 1 to 4 nM showing a peak at 2 nM. Temporal RGCs did not present any significant change in axon growth on EphA3-Fc between 0.5 and 4 nM and presented a significant decrease at 8 nM (ANOVA and Tukey postest, 3 independent experiments, n: 20 longer axons for explant, 3 explants for condition). (E) Quantification of axon length of nasal and temporal explants exposed to soluble clustered Fc or EphA3-Fc at 2 nM. Nasal RGC axons grow significantly longer with EphA3-Fc (ANOVA and Tukey postest, 3 independent experiments, n: 50 longer axons for explant, 3 explants for condition). (F–G) Dissociated nasal retinal neurons immunolabeled against neuron specific βIII tubulin. They present longer axons on clustered EphA3-Fc at 2 nM (G) than on clustered Fc (F). Scale bars  = 10 µm. (H) Quantification of axon length of nasal and temporal dissociated retinal neurons grown on clustered Fc or EphA3-Fc at 2 nM. Nasal retinal neurons grow significantly longer axons on EphA3-Fc. (ANOVA and Tukey postest, 3 independent experiments, n: nasal retinal neurons on EphA3-Fc: 288, nasal retinal neurons on Fc: 328, temporal retinal neurons on EphA3-Fc: 636, temporal retinal neurons on Fc: 646). (I) The plot depicts the distribution of axon length of nasal and temporal dissociated retinal neurons (NRN and TRN) grown on EphA3-Fc versus Fc. Values given on the y-axis indicate the proportion of retinal neurons which axons reach the length shown on the x-axis. Nasal retinal neurons present a higher proportion of axons between 20 and 40 µm (n: nasal retinal neurons on EphA3-Fc: 288, nasal retinal neurons on Fc: 328, temporal retinal neurons on EphA3-Fc: 636, temporal retinal neurons on Fc: 646). Results are shown as mean +/– SE.</p