Neurotrophin and GDNF family ligands promote survival and alter excitotoxic vulnerability of neurons derived from murine embryonic stem cells

Embryonic stem (ES) cells are genetically manipulable pluripotential cells that can be differentiated in vitro into neurons, oligodendrocytes, and astrocytes. Given their potential utility as a source of replacement cells for the injured nervous system and the likelihood that transplantation interventions might include co-application of growth factors, we examined the effects of neurotrophin and GDNF family ligands on the survival and excitotoxic vulnerability of ES cell-derived neurons (ES neurons) grown in vitro. ES cells were differentiated down a neural lineage in vitro using the 4-/4+ protocol (Bain et al., Dev Biol 168:342-57, 1995). RT-PCR demonstrated expression of receptors for neurotrophins and GDNF family ligands in ES neural lineage cells. Neuronal expression of GFRalpha1, GFRalpha2, and ret was confirmed by immunocytochemistry. Exposure to 30-100 ng/ml GDNF or neurturin (NRTN) resulted in activation of ret. Addition of NT-3 and GDNF did not increase cell division but did increase the number of neurons in the cultures 7 days after plating. Pretreatment with NT-3 enhanced the vulnerability of ES neurons to NMDA-induced death (100 microM NMDA for 10 min) and enhanced the NMDA-induced increase in neuronal [Ca2+]i, but did not alter expression of NMDA receptor subunits NR2A or NR2B. In contrast, pretreatment with GDNF reduced the vulnerability of ES neurons to NMDA-induced death while modestly enhancing the NMDA-induced increase in neuronal [Ca2+]i. These findings demonstrate that the response of ES-derived neurons to neurotrophins and GDNF family ligands is largely similar to that of other cultured central neurons.     

Intrathecal injection of GDNF and BDNF induces immediate early gene expression in rat spinal dorsal horn

Glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) are potent trophic factors for dorsal root ganglion cells. In addition, these factors are produced in subsets of dorsal root ganglion cells and transported anterogradely to their terminals in the superficial dorsal horn of the spinal cord, where they constitute the only source of GDNF and BDNF. We investigated the effect of 10 mug GDNF and BDNF injected by lumbar puncture on the expression of the immediate early gene (IEG) products c-Fos, c-Jun, and Krox-24 in the adult rat dorsal horn. In the dorsal horn of S1 spinal segments, GDNF and BDNF induced a strong increase in IEG expression, which was most pronounced in laminae I and II (2.9- to 4.5-fold). More distal from the injection site, in the dorsal horn of L1/L2 spinal segments, the increase in IEG expression was less pronounced, suggesting a concentration-dependent effect. In order to explain the effects of intrathecally injected GDNF, we investigated whether lumbo-sacral dorsal horn neurons expressed RET protein, the signal-transducing element of the receptor complex for GDNF. It was found that several of these neurons contained RET immunoreactivity and that some of the RET-labeled neurons had the appearance of nociceptive-specific cells, confirming their presumed role in pain transmission. Additionally, using double-labeling immunofluorescence combined with confocal microscopy, it was found that after intrathecal GDNF injection 35% of c-Fos-labeled cells were also labeled for RET. These results demonstrate that intrathecally administered GDNF and BDNF induce IEG expression in dorsal horn neurons in the adult rat, supposedly by way of their cognate receptors, which are present on these neurons. We further suggest that the endogenous release of GDNF and BDNF, triggered by nociceptive stimuli, is involved in the induction of changes in spinal nociceptive transmission as in various pain states.     

Anatomical basis of glial cell line-derived neurotrophic factor expression in the striatum and related basal ganglia during postnatal development of the rat

There is increasing evidence that glial cell line-derived neurotrophic factor (GDNF) plays a role as a limiting, striatal target-derived neurotrophic factor for dopamine neurons of the substantia nigra pars compacta (SNpc) by regulating the magnitude of the first phase of postnatal natural cell death which occurs in these neurons. While it has been shown that GDNF mRNA is relatively abundant in postnatal striatum, the cellular basis of its expression has been unknown. We therefore used nonradioactive in situ hybridization and immunohistochemistry to examine the cellular basis of GDNF mRNA and protein expression, respectively, in postnatal striatum and related structures. We found that GDNF mRNA is expressed within medium-sized striatal neurons. Expression in glia was not observed. At the protein level, regionally, GDNF expression in striatum was observed in striosomal patches, as previously described. At a cellular level a few neurons were observed, but they do not account for the striosomal pattern. This pattern is predominantly due to GDNF-positive neuropil. Some of this neuropil arises from tyrosine hydroxylase-positive nigro-striatal dopaminergic afferents. Astrocytic processes do not appear to contribute to the striosomal pattern. GDNF-positive fibers are identified not only within intrinsic striatal neuropil, but also in fibers within the major striatal efferent targets: the globus pallidus, the entopeduncular nucleus, and the SN pars reticulata. We conclude that during normal postnatal development, medium-sized neurons are the principal source of GDNF within the striatum.     

The role of glial cell line-derived neurotrophic factor (GDNF) and integrins for invasion and metastasis in human pancreatic cancer cells

BACKGROUND AND OBJECTIVES: It is generally accepted that the malignancy of pancreatic cancer is dependent upon the extent of invasion as well as metastasis. However, the factors and mechanisms are incompletely understood. We investigated whether glial cell lined-derived neurotrophic factor (GDNF) enhances the invasive and adhesive behaviors of pancreatic cancer cells by altering of the expression of integrins. METHODS: The expression of the GDNF receptor in pancreatic cancer cell lines (SW1990 and Capan-2) was confirmed by RT-PCR. Then we determined the expression of integrin subunits and the alteration of their expression by GDNF using flow-cytometric analysis and a cellular enzyme-linked immunosorbent assay (CELISA). Adhesion and invasion assay were performed to investigate whether increased integrin expression affected the interaction between cancer cells and ECM proteins. RESULTS: The GDNF receptor subunits were expressed in pancreatic cancer cells. GDNF enhanced the expression of some of the integrin subunits and increased their adhesive and invasive abilities. The enhanced expression and associated increase in adhesive and invasive abilities were inhibited by blocking the GDNF receptor or the integrin beta1 subunit. CONCLUSION: The enhancement of integrin expression by GDNF signaling through the GDNF receptor strongly influences invasion and adhesion to ECM proteins by pancreatic cancer cells.     

Development of pontine noradrenergic A5 neurons requires brain-derived neurotrophic factor

Pontine noradrenergic A5 neurons play a pivotal role in maturation and regulation of the brainstem respiratory rhythm-generating network. Analysis of newborn brain-derived neurotrophic factor (BDNF)-null mice revealed a marked loss of tyrosine hydroxylase-positive A5 neurons compared to wildtype controls that was rescued by null mutation of the proapoptotic gene Bax. In cultures of the A5 region from E12.5 rat embryos, BDNF significantly increased the number and branching of tyrosine hydroxylase-positive neurons. Immunoneutralization of endogenous glial cell line-derived neurotrophic factor partially inhibited the BDNF-dependent increase in the number of tyrosine hydroxylase-positive cells without affecting neurite number. The A5 nucleus is the first brainstem cell group identified at which BDNF is required in vivo for development of neurons critical for cardiorespiratory control.     

6-羟基多巴胺对帕金森病大鼠GDNF表达影响的实验研究

目的 :研究 6 -羟基多巴胺 (6 - OHDA)诱发的大鼠帕金森 (PD)模型 GNDF表达。方法 :通过脑内立体定向术注射 6 -羟基多巴胺建立大鼠 PD模型。采用免疫组化的方法观察损伤侧脑胶质细胞源性神经营养因子 (GDNF)表达。结果 :6 - OHDA诱发的 PD鼠 GDNF蛋白表达减低 (P <0 . 0 5 )。结论 :神经营养因子表达减少可能是 6 - OHDA诱发的 PD大鼠发病机制之一     

Down-regulation of GFRalpha-1 expression by antisense oligodeoxynucleotide aggravates thermal hyperalgesia in a rat model of neuropathic pain

Glial cell line-derived neurotrophic factor (GDNF) has been hypothesized to play an important role in the modulation of nociceptive signals especially during neuropathic pain. The present study examined the expression of GDNF and GFRalpha-1 (the high-affinity receptor of GDNF) in dorsal root ganglions (DRG) in a rat model of neuropathic pain induced by chronic constriction injury (CCI) to the sciatic nerve. In order to address the role of GDNF and GFRalpha-1 in neuropathic pain, antisense oligodeoxynucleotide (ODN) specifically against GFRalpha-1 was intrathecally administered to result in down-regulation of GFRalpha-1 expression. The results showed that both the protein and mRNA levels of GDNF and GFRalpha-1 were significantly increased after CCI, while the thermal hyperalgesia of neuropathic pain rats could be significantly aggravated by antisense ODN treatment, but not by normal saline (NS) or mismatch ODN treatment. The present study demonstrated that endogenous GDNF and GFRalpha-1 might play an anti-hyperalgesic role in neuropathic pain of rats. In addition, we found a down-regulation of somatostatin (SOM) in DRG and spinal dorsal horn after expression of GFRalpha-1 was knocked down, which suggested the possible relationship between the anti-hyperalgesic effect of GDNF and GFRalpha-1 on neuropathic pain and endogenous SOM.     

A dynamic regulation of GDNF-family receptors correlates with a specific trophic dependency of cranial motor neuron subpopulations during development

Glial cell line-derived neurotrophic factor (GDNF) family ligands promote the survival of developing motor neurons in vivo and in vitro. However, not all neurons survive with any single ligand in culture and GDNF null mutant mice display only a partial motor neuron loss. An interesting possibility is that subpopulations of motor neurons based on their function and/or their myotopic organization require distinct members of GDNF family ligands. Because responsiveness to the different ligands depends on the expression of their cognate ligand-binding receptor we have herein addressed this issue by examining the expression of GDNF-family receptors (gfr) during development and in the adult in cranial motor nuclei subpopulations. We have furthermore examined the in vivo role of GDNF for cranial motor neuron subpopulations. The shared ret receptor was expressed in all somatic, branchial and visceral cranial embryonic motor nuclei examined, showing that they are all competent to respond to GDNF family ligands during development. At early stages of development both the GDNF receptor, gfralpha1, and the neurturin (NTN) receptor, gfralpha2, were expressed in the oculomotor, facial and spinal accessory, and only gfralpha1 in the trochlear, superior salivatory, trigeminal, hypoglossal and weakly in the dorsal motor nucleus of the vagus and the ambiguous nucleus. The abducens nucleus was negative for both gfralpha1 and gfralpha2. The artemin (ART) receptor, gfralpha3, was expressed only in the superior salivatory nucleus. A motor neuron subnuclei-specific expression of gfralpha1 and gfralpha2 was seen in the facial and trigeminal nuclei which corresponded to their dependence on GDNF in null mutant mice. We found that the expression was dynamic in these nuclei, which may reflect developmental changes in their trophic factor dependency. Analysis of GDNF null mutant mice revealed that the dynamic receptor expression is regulated by the ligand in vivo, indicating that the attainment of changes in dependency could be ligand induced. Our results indicate that specific GDNF family ligands support selective muscle-motor neuron circuits during development.     

Distinct roles for GFRalpha1 and GFRalpha2 signalling in different cranial parasympathetic ganglia in vivo

Neurturin (NRTN), signalling via the GDNF family receptor alpha2 (GFRalpha2) and Ret tyrosine kinase, has recently been identified as an essential target-derived factor for many parasympathetic neurons. NRTN is expressed in salivary and lacrimal glands, while GFRalpha2 and Ret are expressed in the corresponding submandibular, otic and sphenopalatine ganglia. Here, we have characterized in more detail the role of GDNF and NRTN signalling in the development of cranial parasympathetic neurons and their target innervation. Gfra1 mRNA was expressed at E12 but not in newborn cranial parasympathetic ganglia, while Gfra2 mRNA and protein were strongly expressed in newborn and adult cranial parasympathetic neurons and their projections, respectively. In newborn GFRalpha1- or Ret-deficient mice, where many submandibular ganglion neurons were still present, the otic and sphenopalatine ganglia were completely missing. In contrast, in newborn GFRalpha2-deficient mice, most neurons in all these ganglia were present. In these mice, the loss and atrophy of the submandibular and otic neurons were amplified postnatally, accompanied by complete loss of innervation in some target regions and preservation in others. Surprisingly, GFRalpha2-deficient sphenopalatine neurons, whose targets were completely uninnervated, were not reduced in number and only slightly atrophied. Thus, GDNF signalling via GFRalpha1/Ret is essential in the early gangliogenesis of some, but not all, cranial parasympathetic neurons, whereas NRTN signalling through GFRalpha2/Ret is essential for the development and maintenance of parasympathetic target innervation. These results indicate that GDNF and NRTN have distinct functions in developing parasympathetic neurons, and suggest heterogeneity among and within different parasympathetic ganglia.     

Recombinant adeno-associated virus vector expressing glial cell line-derived neurotrophic factor reduces ischemia-induced damage

To explore the potential of using the recombinant adeno-associated viral (rAAV) vector, expressing glial cell line-derived neurotrophic factor (GDNF) as the gene therapy for stroke, we injected rAAV vectors expressing GDNF (rAAV-GDNF) into the cortex of rats which had been experiencing transient bilateral common carotid artery ligation and right middle cerebral artery ligation for 90 min. GDNF levels in cortical tissues of rAAV-GDNF-injected animals were significantly higher than in the control animals injected with rAAV-expressing lacZ (rAAV-lacZ), indicating that rAAV can deliver and express the GDNF gene in cortical tissues. Triphenyltetrazolium chloride tissue stain analysis revealed that the rAAV-delivered GDNF gene could rescue the brain tissues from ischemia-induced injury. Cortical tissues which received rAAV-GDNF injections had both significantly smaller total volumes of infarction and smaller areas of infarction on each brain slice than those which were injected with rAAV-lacZ. An in situ labeling analysis demonstrated significantly less apoptotic cells in cortical tissues rescued by rAAV-GDNF, indicating prevention of apoptosis as the mechanism of cortical cell protection. Moreover, immunohistochemistry staining of Neu-N indicated that the rescued brain tissues contained the same number of Neu-N-positive neuronal cells as contralateral undamaged brain tissues. This provides strong evidence that cortical neuronal cells can be rescued by GDNF gene therapy. Indeed, these findings show that the rAAV is a potential delivery vector of GDNF gene for the therapy of stroke.