Positive and negative interactions of GDNF, NTN and ART in developing sensory neuron subpopulations, and their collaboration with neurotrophins

Glial cell line-derived neurotrophic factor (GDNF), neurturin (NTN) and neublastin/artemin (ART) are distant members of the transforming growth factor beta family, and have been shown to elicit neurotrophic effects upon several classes of peripheral and central neurons. Limited information from in vitro and expression studies has also substantiated a role for GDNF family ligands in mammalian somatosensory neuron development. Here, we show that although dorsal root ganglion (DRG) sensory neurons express GDNF family receptors embryonically, they do not survive in response to their ligands. The regulation of survival emerges postnatally for all GDNF family ligands. GDNF and NTN support distinct subpopulations that can be separated with respect to their expression of GDNF family receptors, whereas ART supports neurons in populations that are also responsive to GDNF or NTN. Sensory neurons that coexpress GDNF family receptors are medium sized, whereas small-caliber nociceptive cells preferentially express a single receptor. In contrast to brain-derived neurotrophic factor (BDNF)-dependent neurons, embryonic nerve growth factor (NGF)-dependent nociceptive neurons switch dependency to GDNF, NTN and ART postnatally. Neurons that survive in the presence of neurotrophin 3 (NT3) or neurotrophin 4 (NT4), including proprioceptive afferents, Merkel end organs and D-hair afferents, are also supported by GDNF family ligands neonatally, although at postnatal stages they lose their dependency on GDNF and NTN. At late postnatal stages, ART prevents survival elicited by GDNF and NTN. These data provide new insights on the roles of GDNF family ligands in sensory neuron development.     

Intrathecally delivered glial cell line-derived neurotrophic factor produces electrically evoked release of somatostatin in the dorsal horn of the spinal cord

Glial cell line-derived neurotrophic factor (GDNF) is a trophic factor with an established role in sensory neuron development. More recently it has also been shown to support adult sensory neuron survival and exert a neuroprotective effect on damaged sensory neurons. Some adult small-sized dorsal root ganglion (DRG) cells that are GDNF-sensitive sensory neurons express the inhibitory peptide somatostatin (SOM). Thus, we tested the hypothesis that prolonged GDNF administration would regulate SOM expression in sensory neuron cell bodies in the dorsal root ganglia (DRG) and activity-induced release of SOM from axon terminals in the dorsal horn. Continuous intrathecal delivery of GDNF for 11-13 days significantly increased the number of small DRG cells that expressed SOM. Furthermore, GDNF treatment evoked SOM release in the isolated dorsal horn following electrical stimulation of the dorsal roots that was otherwise undetectable in control rats. Conversely capsaicin-induced release of SOM (EC(50) 50 nM) was not modified by GDNF treatment. These results show that GDNF can regulate central synaptic function in SOM-containing sensory neurons.     

GDNF

The identification of novel factors that promote neuronal survival could have profound effects on developing new therapeutics for neurodegenerative disorders. Glial cell line-derived neurotrophic factor (GDNF) is a novel protein purified and cloned based on its marked ability to promote dopaminergic neuronal function. GDNF, now known to be the first identified member of a family of factors, signals through the previously known receptor tyrosine kinase, Ret. Unlike most ligands for receptor tyrosine kinases, GDNF does not bind and activate Ret directly, but requires the presence of GPI-linked coreceptors. There are several coreceptors with differing affinities for the GDNF family members. The profile of coreceptors in a cell may determine which factor preferentially activates Ret. In vivo differences in localization of the GDNF family members, its coreceptors and Ret suggest this ligand/receptor interaction has extensive and multiple functions in the CNS as well as in peripheral tissues. GDNF promotes survival of several neuronal populations both in vitro and in vivo. Dopaminergic neuronal survival and function are preserved by GDNF in vivo when challenged by the toxins MPTP and 6-hydroxydopamine. Furthermore, GDNF improves the symptoms of pharmacologically induced Parkinson's disease in monkeys. Several motor neuron populations isolated in vitro are also rescued by GDNF. In vivo, GDNF protects these neurons from programmed cell death associated with development and death induced by neuronal transection. These experiments suggest that GDNF may provide significant therapeutic opportunities in several neurodegenerative disorders.     

miR-22-3p enhances the intrinsic regenerative abilities of primary sensory neurons via the CBL/p-EGFR/p-STAT3/GAP43/p-GAP43 axis

Spinal cord injury (SCI) is a devastating disease. Strategies that enhance the intrinsic regenerative ability are very important for the recovery of SCI to radically prevent the occurrence of sensory disorders. Epidermal growth factor (EGF) showed a limited effect on the growth of primary sensory neuron neurites due to the degradation of phosphorylated-epidermal growth factor receptor (p-EGFR) in a manner dependent on Casitas B-lineage lymphoma (CBL) (an E3 ubiquitin-protein ligase). MiR-22-3p predicted from four databases could target CBL to inhibit the expression of CBL, increase p-EGFR levels and neurites length via STAT3/GAP43 pathway rather than Erk1/2 axis. EGF, EGFR, and miR-22-3p were downregulated sharply after injury. In vivo miR-22-3p Agomir application could regulate CBL/p-EGFR/p-STAT3/GAP43/p-GAP43 axis, and restore spinal cord sensory conductive function. This study clarified the mechanism of the limited promotion effect of EGF on adult primary sensory neuron neurite and targeting miR-22-3p could be a novel strategy to treat sensory dysfunction after SCI.     

Developmental waves of mechanosensitivity acquisition in sensory neuron subtypes during embryonic development

Somatic sensation relies on the transduction of physical stimuli into electrical signals by sensory neurons of the dorsal root ganglia. Little is known about how and when during development different types of sensory neurons acquire transduction competence. We directly investigated the emergence of electrical excitability and mechanosensitivity of embryonic and postnatal mouse sensory neurons. We show that sensory neurons acquire mechanotransduction competence coincident with peripheral target innervation. Mechanotransduction competence arises in different sensory lineages in waves, coordinated by distinct developmental mechanisms. Sensory neurons that are mechanoreceptors or proprioceptors acquire mature mechanotransduction indistinguishable from the adult already at E13. This process is independent of neurotrophin‐3 and may be driven by a genetic program. In contrast, most nociceptive (pain sensing) sensory neurons acquire mechanosensitive competence as a result of exposure to target‐derived nerve growth factor. The highly regulated process of mechanosensory acquisition unveiled here, reveals new strategies to identify molecules required for sensory neuron mechanotransduction.     

Immunohistochemical expression of two members of the GDNF family of growth factors and their receptors in the olfactory system

The glial cell line-derived (GDNF) family of trophic factors, GDNF, neurturin, persephin and artemin, are known to support the survival and regulate differentiation of many neuronal populations, including peripheral autonomic, enteric and sensory neurons. Members of this family of related ligands bind to specific GDNF family receptor (GFR) proteins, which complex and signal through the Ret receptor tyrosine kinase. We showed previously that GDNF protein was detectable in olfactory sensory neurons (OSNs) in the olfactory neuroepithelium (ON). In this immunohistochemical study, we localized GDNF, neurturin, GFRα1, GFRα2 and Ret in the adult rat ON and olfactory bulb. We found that GDNF and Ret were widely expressed by immature and mature OSNs, while neurturin was selectively expressed in a subpopulation of OSNs zonally restricted in the ON. The GFRs had differential expression, with mature OSNs and their axons preferentially expressing GFRα1, whereas progenitors and immature neurons more avidly expressed GFRα2. In the bulb, GDNF was highly expressed by the mitral and tufted cells, and by periglomerular cells, and its distribution generally resembled that of Ret, with the exception that Ret was far more predominant on fibers than cell bodies. Neurturin, in contrast, was present at lower levels and was more restricted in its expression to the axonal compartment. GFRα2 appeared to be the dominant accessory protein in the bulb. These data are supportive of two members of this neurotrophic family, GDNF and neurturin, playing different physiological roles in the olfactory neuronal system.     

GDNF promotes neurite outgrowth and upregulates galectin-1 through the RET/PI3K signaling in cultured adult rat dorsal root ganglion neurons

Galectin-1 (GAL-1), a member of a family of β-galactoside binding animal lectins, is predominantly expressed in isolectin B4 (IB4)-binding small non-peptidergic (glial cell line-derived neurotrophic factor (GDNF)-responsive) sensory neurons in the sections of adult rat dorsal root ganglia (DRG), but its functional role and the regulatory mechanisms of its expression in the peripheral nervous system remain unclear. In the present study, both recombinant nerve growth factor (NGF) and GDNF (50 ng/ml) promoted neurite outgrowth from cultured adult rat DRG neurons, whereas GDNF, but not NGF, significantly increased the number of IB4-binding neurons and the relative protein expression of GAL-1 in the neuron-enriched culture of DRG. The GAL-1 expression in immortalized adult rat Schwann cells IFRS1 and DRG neuron-IFRS1 cocultures was unaltered by treatment with GDNF, which suggests that GDNF/GAL-1 signaling axis is more related to neurite outgrowth, rather than neuron-Schwann cell interactions. The GDNF-induced neurite outgrowth and GAL-1 upregulation were attenuated by anti-GDNF family receptor (RET) antibody and phosphatidyl inositol-3′-phosphate-kinase (PI3K) inhibitor LY294002, suggesting that the neurite-outgrowth promoting activity of GDNF may be attributable, at least partially, to the upregulation of GAL-1 through RET-PI3K pathway. On the contrary, no significant differences were observed between GAL-1 knockout and wild-type mice in DRG neurite outgrowth in the presence or absence of GDNF. Considerable immunohistochemical colocalization of GAL-3 with GAL-1 in DRG sections and GDNF-induced upregulation of GAL-3 in cultured DRG neurons imply the functional redundancy between these galectins.     

Differential Effects of Glial Cell Line-Derived Neurotrophic Factor and Neurturin on Developing and Adult Substantia Nigra Dopaminergic Neurons

Neurturin (NTN) and glial cell line-derived neurotrophic factor (GDNF), two members of the GDNF family of growth factors, exert very similar biological activities in different systems, including the substantia nigra. Our goal in the present work was to compare their function and define whether nonoverlapping biological activities on midbrain dopaminergic neurons exist. We first found that NTN and GDNF are differentially regulated during postnatal development. NTN mRNA progressively decreased in the ventral mesencephalon and progressively increased in the striatum, coincident with a decrease in GDNF mRNA expression. This finding suggested distinct physiological roles for each factor in the nigrostriatal system. We therefore examined their function in ventral mesencephalon cultures and found that NTN promoted survival comparable with GDNF, but only GDNF induced sprouting and hypertrophy of developing dopaminergic neurons. We subsequently examined the ability of NTN to prevent the 6-hydroxydopamine-induced degeneration of adult dopaminergic neurons in vivo. Fibroblasts genetically engineered to deliver high levels of GDNF or NTN were grafted supranigrally. NTN was found to be as potent as GDNF at preventing the death of nigral dopaminergic neurons, but only GDNF induced tyrosine hydroxylase staining, sprouting, or hypertrophy of dopaminergic neurons. In conclusion, our results show selective survival-promoting effects of NTN over wider survival, neuritogenic, and hypertrophic effects of GDNF on dopaminergic neurons in vitro and in vivo. Such differences are likely to underlie unique roles for each factor in postnatal development and may ultimately be exploited in the treatment of Parkinson's disease.     

胶质细胞源性神经营养因子对疼痛的调节作用

胶质细胞源性神经营养因子(glial cell derived neurotrophic factor,GDNF)属转化生长因子β超家族成员,其成熟蛋白由134个氨基酸残基组成,而GDNF受体广泛分布于外周和中枢神经系统。GDNF不仅可以促进多巴胺能神经元、运动神经元的存活,对交感、副交感以及感觉神经元具有营养作用,还能够影响神经元的发育、分化并对非神经系统的发育也具有重要作用。近年来随着人们对疼痛认识的深入,疼痛的机制也不再限于神经元功能的改变,还受胶质细胞活化、多种营养因子、细胞因子及相应受体、离子通道等多方面因素的影响。为此,本文就近年来GDNF参与疼痛调节的相关研究进展做一简要综述。     

BMP‐2 and cAMP elevation confer locus coeruleus neurons responsiveness to multiple neurotrophic factors

The locus coeruleus (LC) is a major target of several neurodegenerative disorders, including Parkinson's and Alzheimer's diseases. However, very little is known of the trophic requirements of LC neurons. In the present work, we have studied the biological activity of neurotrophic factors from different families in E15 primary cultures of LC neurons. In agreement with previous results, neurotrophin‐3 (NT‐3) and also glial cell line‐ derived neurotrophic factor (GDNF) increased the number of embryonic LC noradrenergic neurons in the presence of serum. In serum‐free conditions, none of the factors tested, including NT‐3, GDNF, neurturin, basic fibroblast growth factor (bFGF), or bone morphogenetic protein‐2 (BMP‐2), promoted the survival of tyrosine hydroxylase (TH)‐immunoreactive neurons at 6 days in vitro. However, when BMP‐2 was coadministered with any of these factors the number of LC TH‐positive neurons increased twofold. Similar results were obtained by cotreatment of LC neurons with forskolin and NT‐3, bFGF, or BMP‐2. The strongest effect (a fourfold increase in the number of TH‐positive cells) was induced by cotreatment with forskolin, BMP‐2, and GDNF. Thus, our results show that LC neurons require multiple factors for their survival and development, and suggest that activation of LC neurons by bone morphogenetic proteins and cAMP plays a decisive role in conferring noradrenergic neuron responsiveness to several trophic factors. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 291–304, 2002; DOI 10.1002/neu.10034     

Neurotrophin-4: a survival factor for adult sensory neurons

The nerve growth factor (NGF) family of neurotrophins provides a substantial part of the normal trophic support for sensory neurons during development. Although these neurotrophins, which include Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT-3), and Neurotrophin-4 (NT-4), continue to be expressed into adulthood, there is little evidence that they are survival factors for adult neurons. Here we have examined the age-dependent neurotrophic requirements of a specialized type of mechanoreceptive neuron, called a D-hair receptor, in the dorsal root ganglion (DRG). Studies using knockout mice have demonstrated that the survival of D-hair receptors is dependent upon both NT-3 and NT-4. Here, we show that the time period when D-hair receptors require these two neurotrophins is different. Survival of D-hair receptors depends on NT-3 early in postnatal development and NT-4 later in the mature animal. The age-dependent loss of D-hair neurons in older NT-4 knockout mice was accompanied by a large reduction (78%) in neurons positive for the NT-4 receptor (trkB) together with neuronal apoptosis in the DRG. This is the first evidence that sensory neurons have a physiological requirement for a single neurotrophin for their continued survival in the adult.     

Expression patterns of neurotrophic factor mRNAs in developing human teeth

Neurotrophic factors regulate survival, differentiation, growth and plasticity in the nervous system. In addition, based on their specific and shifting temporospatial expression patterns, neurotrophic factors have been implicated in morphogenetic events during tooth development in rodents. To determine whether these findings in rodents could be related to humans, we have now studied nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), glial cell-line derived neurotrophic factor (GDNF), and neurturin (NTN) mRNA expression patterns in developing human teeth during gestational weeks 6.5-11. Using in situ hybridization histochemistry, we found distinct and specific patterns of neurotrophin and GDNF mRNA expression in the developing human teeth. NGF mRNA labeling was weak and confined predominantly to the dental papilla. BDNF mRNA labeling was stronger than NGF mRNA and was seen in the mesenchyme located lateral to the dental organ, as well as in epithelial structures (inner dental epithelium and enamel knot). NT-3 mRNA was observed in the dental papilla and in the area of the cervical loop. NT-4 mRNA was expressed in both oral and dental epithelia in all stages studied. GDNF mRNA was found in the dental follicle and at different sites in the inner dental epithelium. Weak NTN mRNA labeling was also found in the developing teeth. Based on these findings, we suggest that neurotrophins, GDNF and NTN might be involved in morphogenetic events during early stages of tooth development in humans. Protein gene product (PGP) 9.5-immunoreactive nerve fibers were observed in the dental follicle by 11 weeks coinciding with the labeling for neurotrophic factor mRNAs in this structure. This suggests that these neurotrophic factors might be involved in the innervation of dental structures. The rich expression of neurotrophic factors in developing dental tissues suggests that developing, or possibly adult, dental tissue might be used as an allograft source of trophic support for diseases of the nervous system.     

Nerve growth factor induces P2X(3) expression in sensory neurons

Glial cell line-derived neurotrophic factor (GDNF) and nerve growth factor (NGF) are neuroprotective for subpopulations of sensory neurons and thus are candidates for pain treatment. However, delivering these factors to damaged neurons will invariably result in undamaged systems also being treated, with possible consequences for sensory processing. In sensory neurons the purinergic receptor P2X(3) is found predominantly in GDNF-sensitive nociceptors. ATP signalling via the P2X(3) receptor may contribute to pathological pain, suggesting an important role for this receptor in regulating nociceptive function. We therefore investigated the effects of intrathecal GDNF or NGF on P2X(3) expression in adult rat spinal cord and dorsal root ganglia (DRG). In control spinal cords, P2X(3) expression was restricted to a narrow band of primary afferent terminals within inner lamina II (II(i)). Glial cell line-derived neurotrophic factor treatment increased P2X(3) immunoreactivity within lamina II(i) but not elsewhere in the cord. Nerve growth factor treatment, however, induced novel P2X(3) expression, with intense immunoreactivity in axons projecting to lamina I and outer lamina II and to the ventro-medial afferent bundle beneath the central canal. In the normal DRG, we found a greater proportion of P2X(3)-positive neurons at cervical levels, many of which were large-diameter and calcitonin gene-related peptide-positive. In both cervical and lumbar DRG, the number of P2X(3)-positive cells increased following GDNF or NGF treatment. De novo expression of P2X(3) in NGF-sensitive nociceptors may contribute to chronic inflammatory pain.     

神经营养因子与神经干细胞

生长因子在神经干细胞的增殖,分化和存活过程中有重要作用。神经营养因子是其中的一类,它包括神经生长因子（NGF）家族,胶质源性神经营养因子（GDNF）家族和其它神经营养因子。NGF家族包括NGF,BDNF,NT－3,NT－4／5和NT－6。这一家族可促进epidermic growth facter(EGF)反应 海马及前脑室管膜下区神经干细胞的存活和分化。GDNF家族包括GDNF,NTN,PSP和ART。GDNF家族促神经发育的作用主要在外周,它促进肠神经嵴前体细胞的存活和增殖,且对外周感觉神经的发育至关重要。其它生长因子如bFGF和EGF,它们能促进神经干细胞增殖和存活;CNTF和LIF等在神经干细胞的分化中也有重要作用。     

The role of activin in neuropeptide induction and pain sensation

Signals from target tissues play critical roles in the functional differentiation of neuronal cells, and in their subsequent adaptations to peripheral changes in the adult. Sensory neurons in the dorsal root ganglia (DRG) provide an excellent model system for the study of signals that regulate the development of neuronal diversity. DRG have been well characterized and contain both neurons that convey information from muscles about limb position, as well as other neurons that provide sensations from skin about pain information. Sensory neurons involved in pain sensation can be distinguished physiologically and antigenically, and one hallmark characteristic is that these neurons contain neuropeptides important for their functions. The transforming growth factor (TGF) beta family member activin A has recently been implicated in neural development and response to injury. During sensory neuron development, peripheral target tissues containing activin or activin itself can regulate pain neuropeptide expression. Long after development has ceased, skin target tissues retain the capacity to signal neurons about changes or injury, to functionally refine synapses. This review focuses on the role of activin as a target-derived differentiative factor in neural development that has additional roles in response to cutaneous injuries in the adult.     

Glial cell line-derived neurotrophic factor is expressed in penis of adult rat and retrogradely transported in penile parasympathetic and sensory nerves

Glial cell line-derived neurotrophic factor (GDNF), a member of the GDNF family of neurotrophic factors, promotes the survival and function of several neuronal populations in the peripheral and central nervous system. In the present study, expression of GDNF mRNA in the shaft of adult rat penis is demonstrated. In situ hybridization revealed GDNF mRNA expression in cells lying in the narrow zone between the tunica albuginea and the cavernous tissue. Most subtunical cells exhibited immunoreactivity for vimentin and S100 beta, but they did not stain for smooth muscle alpha actin or PGP9.5. This suggests that the GDNF mRNA-expressing cells may have a mesenchymal origin. Also retrograde axonal transport of intracavernously injected 125I-labeled GDNF in penile parasympathetic and sensory neurons is shown. The transport was inhibited by excess unlabeled GDNF, whereas excess cytochrome c had no effect. This is in agreement with the view that the transport was mediated by binding to specific receptors located on axon terminals. In addition, this study demonstrates expression of GDNF family receptor-alpha 3 (GFR alpha 3) mRNA in most adrenergic, but only in a minor part (5.3%) of the penis-projecting adult rat major pelvic ganglion neurons, as well as in almost half (45.6%) of the penile S1 dorsal root ganglion neurons. In conclusion, the present data suggest that GDNF may act as a neurotrophic factor for subpopulations of adult rat penile parasympathetic and sensory neurons.     

Glial cell line-derived neurotrophic factor-induced signaling in Schwann cells

Glial cell line-derived neurotrophic factor (GDNF), a known survival factor for neurons, has recently been shown to stimulate the migration of Schwann cells (SCs) and to enhance myelination. GDNF exerts its biological effects by activating the Ret tyrosine kinase in the presence of glycosylphosphatidylinositol-linked receptor, GDNF family receptor (GFR) alpha1. In Ret-negative cells, the alternative transmembrane coreceptor is the 140-kDa isoform of neural cell adhesion molecule (NCAM) associated with a non-receptor tyrosine kinase Fyn. We confirmed that GDNF, GFRalpha1 and NCAM are expressed in neonatal rat SCs. We found that GDNF induces an increase in the partitioning of NCAM and heparan sulfate proteoglycan agrin into lipid rafts and that heparinase inhibits GDNF-signaling in SCs. In addition to activation of extracellular signal-regulated kinases, and phosphorylation of cAMP response element binding protein, we found that cAMP-dependent protein kinase A and protein kinase C are involved in GDNF-mediated signaling in SCs. Although GDNF did not promote the differentiation of purified SCs into the myelinating phenotype, it enhanced myelination in neuron-SC cocultures. We conclude that GDNF utilizes NCAM signaling pathways to regulate SC function prior to myelination and at early stages of myelin formation.     

周围神经损伤后外源性GDNF对神经元的保护作用

采用硅管套接大鼠切断的坐骨神经模型 ,局部给予胶质细胞源性神经营养因子 (GDNF) ,应用尼氏染色、酶组织化学染色方法 ,观察到外源性GDNF能减少脊髓修复侧前角运动神经元死亡的数目 ,降低脊髓前角运动神经元及脊神经节感觉神经元中胆碱酯酶 (CHE)及酸性磷酸酶 (ACP)变化的幅度。这表明外源性GDNF能保护周围神经切断后引起的神经元损伤。