The enteric nervous system (ENS) continues its development after birth, with formation of ganglia and functional synapses; plasticity is also demonstrated in significant axon growth that occurs after experimental colitis in the adult colon. However, little is known about factors in the postnatal intestine that influence and regulate these processes. Therefore we tested the effects of known neurotrophins, NGF, NT-3, BDNF and GDNF on neonatal rat myenteric neurons. Cocultures were developed by isolating the myenteric plexus and surrounding muscular wall from neonatal rats, and effects of exogenous treatment of neurotrophins were analyzed using immunocytochemistry and image analysis. Western blotting and immunocytochemistry were performed to detect implicated neurotrophins and their receptors in the postnatal intestine. Functional aspects of effects of implicated neurotrophins were assessed by [3H]choline uptake and acetylcholine release in myenteric neurons. Last, TNBS-colitis was induced in adult rats to determine changes in GDNF secretion during the course of the disease. Application of 100ng/mL GDNF to a neonatal intestinal coculture containing neurons, glia and smooth muscle cells produced a 91.5% (p≤0.05) increase in axons. GDNF induced morphological changes in the structure and organization of neurons and axons; the incidence of neurons present in ganglia increased by 11.2% (p≤0.05), with a 32.9% (p≤0.05) increase in aggregated axons. Western blotting and immunocytochemistry confirmed intestinal smooth muscle as the major source of GDNF and demonstrated the presence of the GDNF receptor complex, GFRα1 and RET in the myenteric plexus. Choline uptake significantly increased at 50, 100 and 150ng/ml doses of GDNF, whereas stimulated ACh release increased only at 100 and 150ng/ml doses. In TNBS-colitis, a decrease in 35kD GDNF at days 1 and 6 post-induction of inflammation was observed, with a concomitant increase in 15kD GDNF. We conclude that GDNF, produced by intestinal smooth muscle, is a key factor influencing development of the postnatal myenteric neuron and may play a role in ENS-restructuring post-inflammation. / Thesis (Master, Physiology) -- Queen's University, 2008-09-03 13:27:23.042

According to the literature, there is a decrease in glial cell number or hypofunction of glial cells in depression. It was also found that both antidepressants and atypical antipsychotics might target glial cells, and that they increase the release of glial-cell-line-derived neurotrophic factor (GDNF) from C6 rat glioma cells (C6 cells). In this project, C6 cells were used as a model for glial cells to investigate the effects of fluoxetine and quetiapine on proliferation and differentiation, and to investigate their effects on the release of GDNF. A combination of quetiapine and fluoxetine was used to study their potential synergistic effect on the release of GDNF from C6 cells. <p>C6 cells were treated with different concentrations of fluoxetine and quetiapine in both normal and serum starvation culture conditions. Under the serum present condition, fluoxetine (25 mM) decreased the number of C6 cells from 24 to 48 h, while quetiapine (25 mM) decreased the cell number only at 48h. Under serum starvation, it was found that fluoxetine (12.5 mM) increased the number of C6 cells from 24 to 48 h treatment; in contrast, quetiapine (25 mM) decreased the number of C6 cells after 48 h treatment. Both fluoxetine and quetiapine inhibited the proliferation of C6 cells under normal and serum starvation conditions. Fluoxetine (12.5 mM) decreased C6 cell death, while quetiapine had no significant effect. Fluoxetine, but not quetiapine, changed the morphology of C6 cells and increased the level of glial fibrillary acidic protein (GFAP), an astrocyte marker. Both fluoxetine (12.5, 25 mM) and quetiapine (25 mM) increased the release of GDNF from C6 cells, and an apparent additive effect was found between quetiapine and fluoxetine in the modulation of release of GDNF from these cells. <p>It was concluded that:<p>1. High concentration (25 mM) of fluoxetine and quetiapine decreased the number of C6 cells under the serum present condition and both drugs inhibited the proliferation of C6 cells.<p>2. Fluoxetine had a protective effect on the C6 cells under serum starvation, and affected the differentiation of C6 cells; this implies that fluoxetine may protect glial cells in vivo and affect their differentiation. <p>3. A high concentration of quetiapine decreased the number of C6 cells and inhibited the proliferation under serum starvation; even though it increased the release of GDNF from C6 cells as did fluoxetine.<p>4. Both quetiapine and fluoxetine increased the release of GDNF from <p>C6 cells under serum starvation. The combination of quetiapine and fluoxetine had an apparent additive effect in the modulation of GDNF release.<p>5. These effects on proliferation & GDNF release may underlie the benefit observed with these drugs in treating depression and schizophrenia.

According to the literature, there is a decrease in glial cell number or hypofunction of glial cells in depression. It was also found that both antidepressants and atypical antipsychotics might target glial cells, and that they increase the release of glial-cell-line-derived neurotrophic factor (GDNF) from C6 rat glioma cells (C6 cells). In this project, C6 cells were used as a model for glial cells to investigate the effects of fluoxetine and quetiapine on proliferation and differentiation, and to investigate their effects on the release of GDNF. A combination of quetiapine and fluoxetine was used to study their potential synergistic effect on the release of GDNF from C6 cells. <p>C6 cells were treated with different concentrations of fluoxetine and quetiapine in both normal and serum starvation culture conditions. Under the serum present condition, fluoxetine (25 mM) decreased the number of C6 cells from 24 to 48 h, while quetiapine (25 mM) decreased the cell number only at 48h. Under serum starvation, it was found that fluoxetine (12.5 mM) increased the number of C6 cells from 24 to 48 h treatment; in contrast, quetiapine (25 mM) decreased the number of C6 cells after 48 h treatment. Both fluoxetine and quetiapine inhibited the proliferation of C6 cells under normal and serum starvation conditions. Fluoxetine (12.5 mM) decreased C6 cell death, while quetiapine had no significant effect. Fluoxetine, but not quetiapine, changed the morphology of C6 cells and increased the level of glial fibrillary acidic protein (GFAP), an astrocyte marker. Both fluoxetine (12.5, 25 mM) and quetiapine (25 mM) increased the release of GDNF from C6 cells, and an apparent additive effect was found between quetiapine and fluoxetine in the modulation of release of GDNF from these cells. <p>It was concluded that:<p>1. High concentration (25 mM) of fluoxetine and quetiapine decreased the number of C6 cells under the serum present condition and both drugs inhibited the proliferation of C6 cells.<p>2. Fluoxetine had a protective effect on the C6 cells under serum starvation, and affected the differentiation of C6 cells; this implies that fluoxetine may protect glial cells in vivo and affect their differentiation. <p>3. A high concentration of quetiapine decreased the number of C6 cells and inhibited the proliferation under serum starvation; even though it increased the release of GDNF from C6 cells as did fluoxetine.<p>4. Both quetiapine and fluoxetine increased the release of GDNF from <p>C6 cells under serum starvation. The combination of quetiapine and fluoxetine had an apparent additive effect in the modulation of GDNF release.<p>5. These effects on proliferation & GDNF release may underlie the benefit observed with these drugs in treating depression and schizophrenia.

In der vorliegenden Dissertation wurde die subzelluläre Lokalisation der Rezeptoren für die neurotrophen Faktoren BDNF, CNTF und GDNF in primären embryonalen und adulten Motoneuronen erstmalig genau charakterisiert. Die Rezeptoruntereinheiten des BDNF und CNTF Rezeptors, TrkB, p-TrkB, gp130 und p-Stat3, sind im Perikaryon, in Dendriten, im Axon und an den Axonterminalen bzw. Wachstumskegeln von Motoneuronen lokalisiert. Dabei sind die nativen Formen (TrkB, gp130) im Axon überwiegend membranständig, die aktivierten Formen (p-TrkB, p-Stat3) überwiegend im Inneren des Axons lokalisiert. Demgegenüber sind die Rezeptoruntereinheiten des GDNF Rezeptors, Ret und p-Ret, besonders stark in den Dendriten exprimiert. Auch im Perikaryon und an der neuromuskulären Endplatte sind Ret und p-Ret lokalisiert, nicht jedoch im Axon. Im zweiten Teil der Arbeit wurde das durch neurotrophe Faktoren bedingte Neuritenwachstum genau quantifiziert. Dabei wurde zwischen einer Stimulation des Axon- bzw. des Dendritenwachstums differenziert. Die mit GDNF behandelten Dendriten werden etwa doppelt so lang wie die Dendriten, der mit BDNF oder CNTF behandelten Motoneurone. GDNF ist somit ein potenter Stimulator des Dendritenwachstums bei isolierten primären Motoneuronen. Dieser Befund korreliert gut mit der starken Expression von Ret und p-Ret in den Dendriten. Des Weiteren wurde eine Analyse der Interaktion der neurotrophen Faktoren mit dem glutamatergen AMPA Rezeptor in Hinblick auf das Neuritenwachstum durchgeführt. Dabei zeigte sich, dass die Interaktion zwischen neurotrophen Faktoren und dem AMPA Rezeptor besonders für das Dendritenwachstum von Bedeutung ist. Die klinische Bedeutung neurotropher Faktoren und deren Rezeptoren wird im dritten Teil der Arbeit dargestellt. Die pmn Maus ist ein Mausmodell für humane degenerative Erkrankungen des Motoneurons, wie der ALS und der SMA. Pmn Motoneurone, die mit BDNF oder GDNF kultiviert werden, weisen den charakteristischen axonalen Wachstumsdefekt der pmn Motoneurone auf und werden nur etwa halb so lang wie gesunde Kontrollmotoneurone. Bemerkenswerterweise führt die Behandlung der pmn Motoneurone mit CNTF zu einer kompletten Remission des axonalen Wachstumsdefekts, so dass die Axone eine normale Axonlänge erreichen. Auch die Anzahl der pathologischen axonalen Schwellungen werden in vitro durch CNTF stark reduziert. CNTF scheint demnach der interessanteste neurotrophe Faktor für eine Behandlung degenerativer Motoneuronerkrankungen zu sein. / Neurotrophins are important factors for many different functions of motoneurons, such as survival, neurite growth, as well as neuromuscular signalling. Neurotrophin receptors are therefore thought to be differently distributed in dendrites and axons. However, their precise localization and regulation in motoneurons were not well defined. This thesis characterized the exact subcellular localisation of the BDNF, CNTF and GDNF receptor subunits on adult and embryonic motoneurons. The BDNF und CNTF receptor subunits, gp130 and p-Stat3, are located in the perikaryon, in dendrites, in the axon as well as the growth cones and neuromuscular junctions of motoneurons. Immunofluorescent staining for the native forms (TrkB, gp130) is mainly found close to the membrane of the axon. In contrast, the activated forms (p-TrkB, p-Stat3) are mainly located inside the axon. GDNF receptor subunits Ret and p-Ret are highly expressed in the dendrites of motoneurons. In addition, Ret and p-Ret are also located in the perikaryon as well as the neuromuscular junction. Moreover, neurite outgrowth stimulated by neurotrophic factors was analyzed, differentiating axonal and dendritic growth. Primary motoneurons treated with GDNF grew dendrites which were twice as long as dendrites treated with BDNF or CNTF. Thus, GDNF is an important and potent stimulator of dendrite outgrowth in isolated primary motoneurons. This finding correlates well with the high expression of Ret and p-Ret in dendrites. On the other hand BDNF, CNTF and GDNF had equally potent effects on stimulating axonal growth. This thesis also characterized the interactions of neurotrophic factors with AMPA receptors regarding effects on neurite outgrowth. Interestingly, this interaction seems to be of greater importance for dendritic growth rather than axonal growth. The pmn mouse is a mouse model for neurodegenerative diseases of motoneurons, such as amyotrophic lateral sclerosis and spinal muscular atrophy. Pmn Motoneurons, which were cultured in presence of BDNF or GDNF, displayed the characteristic axonal growth deficiency as well as typical axonal swellings. The axon of these motoneurons reached only half the length of healthy control motoneurons. Surprisingly, treatment with CNTF rescued the pmn phenotype as the axons grew to the lengths of healthy control motoneurons. CNTF treatment also significantly reduced the number of pathological axonal swellings in vitro. Therefore CNTF seems to be the most promising therapeutic neurotrophic factor for treatment of neurodegenerative diseases of the motoneuron.

Neurturin (NTN) belongs to the glial cell-line derived neurotrophic factor (GDNF) family of growth factors. Both NTN and GDNF have been shown to potently prevent the degeneration of dopaminergic neuron in vitro and in vivo. The GDNF family receptor alpha 2 (GFRα-2) is the preferred receptor for NTN. In addition to the known full-length isoform (GFRα-2a), we have previously reported the isolation of two novel alternatively spliced isoforms (GFRα-2b and GFRα-2c). The expression levels of these isoforms have yet to be quantified and the functional properties determined. In this report, we have developed a real time polymerase chain reaction (PCR) using SYBR Green I to detect the expression levels of the three splice variants (GFRα-2a, GFRα-2b and GFRα-2c) in murine tissues. Both GFRα-2a and GFRα-2c were expressed at similar levels in all tissues examined. GFRα-2b was found to be 10 fold lower in expression. All three isoforms activated MAPK (ERK1/2) and Akt. Transcriptional profiling with DNA microarrays demonstrated that the spliced isoforms do not share similar profiles. In conclusion, we have now shown the expression levels of the spliced variants. All three isoforms are functional. However, each isoform appeared to have unique transcriptional profiles when activated. / Singapore-MIT Alliance (SMA)

Damage to peripheral nerves following trauma or disease has a number of consequences including burning pain, muscle wasting, paralysis, or organ dysfunction. The most common form of neuropathy is that associated with metabolic abnormality, notably diabetes. Many diabetics, especially those with poor blood sugar control, ultimately develop a distal symmetrical and painful neuropathy that initially affects the longest peripheral axons, but with time spreads proximally. Deficiency in neurotrophic support has been proposed to contribute to the development of diabetic neuropathy. Recently, peripheral gene delivery of vascular endothelial growth factor (VEGF), neurotrophin-3 (NT-3), NGF, BDNF or hepatocyte growth factor (HGF) has been shown to facilitate the continuous production of neurotrophic factors and alleviate the diabetic neuropathy. The role of glial cell-derived neurotrophic factor (GDNF) in the pathogenesis and therapeutics of diabetic neuropathy is not well defined. The main objectives of this research sought to inspect the protective effect of GDNF peripheral gene delivery during hyperglycemia- or constriction- induced sciatic nerve injury in rats. In present proposal, we propose to investigate the change in organization and expressions of GDNF signaling complex in the sciatic nerve following injury in the initial stage. Subsequently, the recombinant adenovirus was used gene delivery system for GDNF to evaluate the potential of intramuscular administration of gene delivery for prevent nerve degeneration, and the molecular mechanism of GDNF to ameliorate neuropathy will be clarified. The above study would enable us to test the hypothesis that the topical gene delivery might be a suitable strategy for the treatment of diabetic neuropathy and other disorders in peripheral nerve. Furthermore, the results of animal studies might be extrapolated for future clinical application.

No description available.

The glial cell-line derived neurotrophic factor (GDNF) and neurturin (NTN) belong to a structurally related family of neurotrophic factors. NTN exerts its effect through a multi-component receptor system consisting of the GDNF family receptor alpha 2 (GFRα2), proto-oncogene RET and/or NCAM. GFRα2 is spliced into at least three isoforms, GFRα2a, GFRα2b and GFRα2c. The present study investigated the expression and functional differences of GFRα2 isoforms. These receptor isoforms are differentially expressed in specific human brain regions. Using Neuro2A model, GDNF and NTN promote neurite outgrowth via GFRα2a and GFRα2c, but not GFRα2b. These GFRα2 isoforms regulate different early response genes when stimulated with GDNF and NTN. Interestingly, using co-expression models, GFRα2b inhibits ligand induced neurites outgrowth of GFRα2a and GFRα2c, and also the related receptor, GFRα1a. More intriguingly, ligands activated GFRα2b was also able to attenuate neurite extension induced by an unrelated stimulation using retinoic acid. MAPK activation induced by GDNF was not attenuated by GFRα2b in a co-expression model, while the early response genes expression profile (up-regulation of FosB) was similar to that induced by GFRα2b alone. This study suggest that GFRα2b is not merely a dominant negative isoform, but signals through a yet to be determined mechanism to antagonize and inhibit neuritogenesis. Together, these data suggest a new paradigm for the regulation of growth factor signaling and neurite outgrowth via an inhibitory splice variant of the receptor. / Singapore-MIT Alliance (SMA)

A theoretical post-translational processing model of the proprotein form of glial cell line-derived neurotrophic factor (GDNF) likely produces three biologically active peptides. The three prospective peptides formed are 5, 11, and 17 amino acid peptides, entitled dopamine neuron stimulating peptide -5 (DNSP-5), -11 (DNSP-11), and -17 (DNSP-17), respectively. The DNSPs were hypothesized to increase dopaminergic neuron function because of their relationship to GDNF: a molecule with established neurotrophic actions on dopaminergic neurons. The DNSPs have the potential to provide a therapeutic molecule similar to GDNF, but with increased ease of delivery and improved bioavailability. Neurochemical effects of DNSPs were examined in the nigrostriatal pathway of normal Fischer 344 rats, and DNSP-11 was found to be the most effective in increasing dopamine neurochemical function. Striatal microdialysis, four weeks after a single intranigral administration of DNSP-11, showed significant increases in the baseline concentrations of dopamine, DOPAC, and HVA. In addition, both, potassium and d-amphetamine-evoked dopamine overflow were significantly increased. DNSP-11 was delivered intranigrally to aged Fischer 344 rats to examine DNSP-11’s ability to improve dopaminergic function in aged dopamine neurons. DNSP-11 affected striatal dopaminergic function 28 days after treatment by decreasing baseline concentrations of dopamine and evoked dopamine release. Investigation of DNSP-11 continued, using two models of neurotoxin-induced dopamine neuron loss that model cell loss associated with Parkinson’s disease. The neuroprotective properties of DNSP-11 were evaluated by delivering DNSP-11 prior to the neurotoxic insult. DNSP-11 treatment was unable to protect dopaminergic neurons, but significantly increased dopamine metabolism. In a model of severe dopamine neuron loss, DNSP-11 treatment significantly improved apomorphine-induced rotation behavior, indicative of alterations in the function of nigrostriatal dopaminergic neurons. Subsequent examination of dopamine content within the SN revealed significant increases in dopamine and DOPAC levels by DNSP-11. Taken together, DNSP-11 treatments modified dopamine neurochemistry in all investigated rodent models. The observed long-term alterations of dopamine neurochemistry by DNSP-11 and subsequent behavioral changes support a potential use for DNSP-11 as a therapeutic for dopaminergic cell loss. Increased dopaminergic function by DNSP-11 is evidence for the novel concept that peptides contained within the prodomain of trophic factors can have neurotrophic actions.

Stem/progenitor cells are present in the adult brain; they undergo constantproliferation and differentiate into mature neurons in certain brain areas, a phenomenoncalled neurogenesis. This study investigated the effects of GDNF, a potent trophic factorof dopaminergic neurons, on neurogenesis in the brain. Nestin and 5-Bromo-2'-deoxyuridine (BrdU) were used as stem/progenitor cells markers.First, we observed extensive bilateral increases of stem/progenitor cells in thedentate gyrus and substantia nigra after continuous infusion of GDNF into the normal ratbrain. However, none of the BrdU+ cells showed neuronal features in the substantia nigraas characterized by immunocytochemical procedures. Next, we identified themorphology of BrdU+ cells after infusing the marker into the brain. While the proceduresincreased the BrdU labeling, neurogenesis was not observed in the basal ganglia. Underelectron microscope, the BrdU+ cells either were undifferentiated or showedcharacteristics of astrocytes. This observation is consistent with suggestions thatastrocytes serve as multipotent progenitors. Later, we repeated GDNF intrastriatalinfusion one month after a severe 6-hydroxydopamine (6-OHDA) lesion. The number ofBrdU+ cells was significantly higher in the GDNF recipients in the ipsilateral substantianigra and both sides of the dentate gyrus. However, no neurogenesis was observed. Inaddition, motor functions were not improved by GDNF treatment. Thus, we measured theeffects of GDNF administration directly into the substantia nigra six hours before apartial 6-OHDA lesion. HPLC measurements of dopamine and its metabolites showed asignificant increase of tissue level in the substantia nigra and striatum, respectively.Despite this, no newly generated dopaminergic neurons was detected in the basal ganglia.Taken together, our studies investigated the effects of GDNF on adultstem/progenitor cells in normal and lesioned rat brain. For the first time, we demonstratedthat GDNF promoted their proliferation in the dentate gyrus, suggesting it has a role inneurogenesis and the function of learning and memory. In each scenario, GDNFpromoted stem/progenitor cell proliferation, but failed to induce neurogenesis in thesubstantia nigra. We believed that the local microenvironment in the substantia nigra mayprevent the stem/progenitor cells to mature into functional neurons.