Positive autoregulation of GDNF levels in the ventral tegmental area mediates long-lasting inhibition of excessive alcohol consumption.

Glial cell line-derived neurotrophic factor (GDNF) is an essential growth factor for the survival and maintenance of the midbrain dopaminergic (DA-ergic) neurons. Activation of the GDNF pathway in the ventral tegmental area (VTA), where the GDNF receptors are expressed, produces a long-lasting suppression of excessive alcohol consumption in rats. Previous studies conducted in the DA-ergic-like cells, SHSY5Y, revealed that GDNF positively regulates its own expression, leading to a long-lasting activation of the GDNF signaling pathway. Here we determined whether GDNF activates a positive autoregulatory feedback loop in vivo within the VTA, and if so, whether this mechanism underlies the long-lasting suppressive effects of the growth factor on excessive alcohol consumption. We found that a single infusion of recombinant GDNF (rGDNF; 10 μg) into the VTA induces a long-lasting local increase in GDNF mRNA and protein levels, which depends upon de novo transcription and translation of the polypeptide. Importantly, we report that the GDNF-mediated positive autoregulatory feedback loop accounts for the long-lasting inhibitory actions of GDNF in the VTA on excessive alcohol consumption. Specifically, the long-lasting suppressive effects of a single rGDNF infusion into the VTA on excessive alcohol consumption were prevented when protein synthesis was inhibited, as well as when the upregulation of GDNF expression was prevented using short hairpin RNA to focally knock down GDNF mRNA in the VTA. Our results could have implications for the development of long-lasting treatments for disorders in which GDNF has a beneficial role, including drug addiction, chronic stress and Parkinson's disease

Glial cell line-derived neurotrophic factor influences proliferation of osteoblastic cells

Little is known about the role of neurotrophic growth factors in bone metabolism. This study investigated the short-term effects of glial cell line-derived neurotrophic factor (GDNF) on calvarial-derived MC3T3-E1 osteoblasts. MC3T3-E1 expressed GDNF as well as its canonical receptors, GFRα1 and RET. Addition of recombinant GDNF to cultures in serum-containing medium modestly inhibited cell growth at high concentrations; however, under serum-free culture conditions GDNF dose-dependently increased cell proliferation. GDNF effects on cell growth were inversely correlated with its effect on alkaline phosphatase (ALP) activity showing a significant dose-dependent inhibition of relative ALP activity with increasing concentrations of GDNF in serum-free culture medium. Live/dead and lactate dehydrogenase assays demonstrated GDNF did not significantly affect cell death or survival under serum-containing and serum-free conditions. The effect of GDNF on cell growth was abolished in the presence of inhibitors to GFR α 1 and RET indicating that GDNF stimulated calvarial osteoblasts via its canonical receptors. Finally, this study found that GDNF synergistically increased tumor necrosis factor-α (TNF-α)-stimulated MC3T3-E1 cell growth suggesting that GDNF interacted with TNF-α-induced signaling in osteoblastic cells. In conclusion, this study provides evidence for a direct, receptor-mediated effect of GDNF on osteoblasts highlighting a novel role for GDNF in bone physiology. \ud \u

Does GDNF exert its neuroprotective effects on photoreceptors in the rd1 retina through the glial glutamate transporter GLAST?

PURPOSE: We previously demonstrated that exogenous glial cell line-derived neurotrophic factor (GDNF) induces histological and functional protection of photoreceptors in the retinal degeneration (rd1) mouse model. The mechanisms underlying such neuroprotection remain elusive. In parallel to this work, we provided evidence for the occurrence of glutamate-mediated excitotoxic phenomena contributing to rod photoreceptor death in the rd1 retina in the companion paper. In the present study, we investigated whether, as demonstrated in other models, GDNF could exert its neuroprotective effect on photoreceptors through Muller glial cells (MGC) by promoting the expression of the glial L-glutamate/L-aspartate transporter (GLAST), an endogenous neuroprotective mechanism against glutamate-mediated excitotoxicity. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was used to compare the mRNA expression levels of GDNF receptors between rd1 and wild-type mouse retinas as well as between MGC and mixed retinal cell cultures. Recombinant GDNF was applied to pure MGC cultures, to rd1 retinal organ cultures and injected subretinally into rd1 mouse eyes. GLAST expression following GDNF treatment was measured by RT-PCR, immunoblotting and immunohistochemistry. Free glutamate and glutamine levels were quantified in rd1 retinas after GDNF or control treatment using an amino acid analyzer. RESULTS: mRNA expression studies of GDNF receptors, GFRalpha-1 and Ret, demonstrated that GDNF receptors were not exclusively expressed by the degenerating photoreceptor cells but mainly by MGC. Exogenous GDNF application to MGC cultures, rd1 mouse retinal explants and in vivo rd1 mouse retinas increased the expression of GLAST by 48% in retinal explants (p<0.005) and by 25% in vivo (p<0.0005). GLAST protein expression in MGC was particularly increased around degenerative photoreceptors. Free glutamate and glutamine levels in the rd1 retina were not significantly modified by exogenous GDNF. CONCLUSIONS: Our data suggest that, in the rd1 mouse retina, GDNF neuroprotective effect on photoreceptors can be mediated indirectly through the activation of MGC. We demonstrate that injection of recombinant GDNF enhances the expression of GLAST and more particularly around the degenerating photoreceptors. Since we failed to demonstrate that GDNF decreases free glutamate levels, we could not ascertain whether GDNF promoted photoreceptor-survival via an increase of glutamate uptake and, therefore, a change in glutamate distributio

Caspase-independent programmed cell death triggers Ca2PO4 deposition in an in vitro model of nephrocalcinosis

We provide evidence of caspase-independent cell death triggering the calcification process in GDNF-silenced HK-2 cells

Dynamic Image-Based Modelling of Kidney Branching Morphogenesis

Kidney branching morphogenesis has been studied extensively, but the mechanism that defines the branch points is still elusive. Here we obtained a 2D movie of kidney branching morphogenesis in culture to test different models of branching morphogenesis with physiological growth dynamics. We carried out image segmentation and calculated the displacement fields between the frames. The models were subsequently solved on the 2D domain, that was extracted from the movie. We find that Turing patterns are sensitive to the initial conditions when solved on the epithelial shapes. A previously proposed diffusion-dependent geometry effect allowed us to reproduce the growth fields reasonably well, both for an inhibitor of branching that was produced in the epithelium, and for an inducer of branching that was produced in the mesenchyme. The latter could be represented by Glial-derived neurotrophic factor (GDNF), which is expressed in the mesenchyme and induces outgrowth of ureteric branches. Considering that the Turing model represents the interaction between the GDNF and its receptor RET very well and that the model reproduces the relevant expression patterns in developing wildtype and mutant kidneys, it is well possible that a combination of the Turing mechanism and the geometry effect control branching morphogenesis

The GDNF-GFRα1 complex promotes the development of hippocampal dendritic arbors and spines via NCAM

The formation of synaptic connections during nervous system development requires the precise control of dendrite growth and synapse formation. Although glial cell line-derived neurotrophic factor (GDNF) and its receptor GFRα1 are expressed in the forebrain, the role of this system in the hippocampus remains unclear. Here, we investigated the consequences of GFRα1 deficiency for the development of hippocampal connections. Analysis of conditional Gfra1 knockout mice shows a reduction in dendritic length and complexity, as well as a decrease in postsynaptic density specializations and in the synaptic localization of postsynaptic proteins in hippocampal neurons. Gain- and loss-of-function assays demonstrate that the GDNF-GFRα1 complex promotes dendritic growth and postsynaptic differentiation in cultured hippocampal neurons. Finally, in vitro assays revealed that GDNF-GFRα1- induced dendrite growth and spine formation are mediated by NCAM signaling. Taken together, our results indicate that the GDNF-GFRα1 complex is essential for proper hippocampal circuit development.Fil: Irala, Dolores. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Bonafina, Antonela. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Fontanet, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Alsina, Fernando Cruz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Paratcha, Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Ledda, Maria Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentin

Enhancement of HGF-induced tubulogenesis by endothelial cell-derived GDNF

学位記番号：医博甲175

Pre-α-pro-GDNF and Pre-β-pro-GDNF Isoforms Are Neuroprotective in the 6-hydroxydopamine Rat Model of Parkinson's Disease

Glial cell line-derived neurotrophic factor (GDNF) is one of the most studied neurotrophic factors. GDNF has two splice isoforms, full-length pre-alpha-pro-GDNF (u-GDNF) and pre-beta-pro-GDNF (beta-GDNF), which has a 26 amino acid deletion in the pro-region. Thus far, studies have focused solely on the u-GDNF isoform, and nothing is known about the in vivo effects of the shorter beta-GDNF variant. Here we compare for the first time the effects of overexpressed cx-GDNF and beta-GDNF in non-lesioned rat striatum and the partial 6-hydroxydopamine lesion model of Parkinson's disease. GDNF isoforms were overexpressed with their native pre-pro-sequences in the striatum using an adeno-associated virus (AAV) vector, and the effects on motor performance and dopaminergic phenotype of the nigrostriatal pathway were assessed. In the non-lesioned striatum, both isoforms increased the density of dopamine transporter-positive fibers at 3 weeks after viral vector delivery. Although both isoforms increased the activity of the animals in cylinder assay, only u-GDNF enhanced the use of contralateral paw. Four weeks later, the striatal tyrosine hydroxylase (TH)-immunoreactivity was decreased in both u-GDNF and 1-GDNF treated animals. In the neuroprotection assay, both GDNF splice isoforms increased the number of TH-immunoreactive cells in the substantia nigra but did not promote behavioral recovery based on amphetamine-induced rotation or cylinder assays. Thus, the shorter GDNF isoform, beta-GDNF, and the full-length alpha-isoform have comparable neuroprotective efficacy on dopamine neurons of the nigrostriatal circuitry.Peer reviewe