GDNF Family Ligands Research Articles

Normalizing GDNF expression in the spinal cord alleviates cutaneous hyperalgesia but not ongoing pain in a rat model of bone cancer pain.

Bone cancer pain (BCP) is the most common complication in patients with bone cancer. Glial cell line-derived neurotrophic factor (GDNF) is believed to be involved in chronic pain conditions. In this article, the expression and roles of GDNF were studied in a rat model of BCP induced by tibia injection of Walker 256 rat mammary gland carcinoma cells. Significant mechanical and thermal hyperalgesia and ongoing pain were observed beginning as early as day 5 post injection. The expression level of GDNF protein examined on day 16 after tibia injection was decreased in the L3 dorsal root ganglion (DRG) and lumbar spinal cord, but not in other spinal levels or the anterior cingulate cortex. Phosphorylation of Ret, the receptor for GDNF family ligands, was also decreased. Furthermore, normalizing GDNF expression with lentiviral vector constructs in the spinal cord significantly reduced mechanical and thermal hyperalgesia, spinal glial activation, and pERK induction induced by tibia injection, but did not affect ongoing pain. Together these findings provide new evidence for the use of GDNF as a therapeutic treatment for bone cancer pain states.

Glial cell line-derived neurotrophic factor family ligands and their therapeutic potential

Four glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) have been characterized: GDNF, neurturin (NRTN), artemin (ARTN) and persephin (PSPN). These proteins support and restore multiple neuronal populations such as dopaminergic, sensory, motor, hippocampal, basal forebrain, enteric, sympathetic and parasympathetic neurons. Therefore, GFLs attracted significant attention as a potential cure for the diseases caused by neuronal injury and degeneration. Results of multiple experiments indicate that GFLs can alleviate behavioral symptoms and restore affected neurons in animal models of several neurological disorders including, among others, Parkinson's disease (PD). During the last decade, GDNF protein and NRTN gene therapy have been tested in several clinical trials in patients with PD. Although the results of phase I clinical trials were positive, phase II clinical trials failed to reach primary end-points. Poor pharmacokinetic properties of GFLs (inability to penetrate tissues barriers, high affinity for extracellular matrix, etc.) could contribute to the absence of clear clinical benefits of these proteins for the patients. The purpose of this paper was to review therapeutic potential of GFLs and discuss possibilities to overcome difficulties associated with pharmacokinetic properties and delivery of GFLs to target neurons.

Neurite outgrowth in normal and injured primary sensory neurons reveals different regulation by nerve growth factor (NGF) and artemin.

Neurotrophic factors have been intensively studied as potential therapeutic agents for promoting neural regeneration and functional recovery after nerve injury. Artemin is a member of the glial cell line-derived neurotrophic factor (GDNF) family of ligands (GFLs) that forms a signalling complex with GFRα3 and the tyrosine kinase Ret. Systemic administration of artemin in rodents is reported to facilitate regeneration of primary sensory neurons following axotomy, improve recovery of sensory function, and reduce sensory hypersensitivity that is a cause of pain. However, the biological mechanisms that underlie these effects are mostly unknown. This study has investigated the biological significance of the colocalisation of GFRα3 with TrkA (neurotrophin receptor for nerve growth factor [NGF]) in the peptidergic type of unmyelinated (C-fibre) sensory neurons in rat dorsal root ganglia (DRG). In vitro neurite outgrowth assays were used to study the effects of artemin and NGF by comparing DRG neurons that were previously uninjured, or were axotomised in vivo by transecting a visceral or somatic peripheral nerve. We found that artemin could facilitate neurite initiation but in comparison to NGF had low efficacy for facilitating neurite elongation and branching. This low efficacy was not increased when a preconditioning in vivo nerve injury was used to induce a pro-regenerative state. Neurite initiation was unaffected by artemin when PI3 kinase and Src family kinase signalling were blocked, but NGF had a reduced effect.

The effect of lentivirus-mediated PSPN genetic engineering bone marrow mesenchymal stem cells on Parkinson's disease rat model.

Persephin (PSPN) is one of the neurotrophic factors of the glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) which have been found to promote the survival of specific populations of neurons. The aim of this study was to assess the potential therapeutic function of gene-modified mesenchymal stem cells (MSCs)-Lv-PSPN-MSCs in 6-OHDA-induced Parkinson’s disease (PD) rats models. Here, we worked on the isolation, purification, identification and amplification of MSCs in vitro. The expression analysis revealed that several of the neural marker proteins like nestin, GFAP and S100 were expressed by rat MSCs. MES23.5 cells co-cultured with Lv-PSPN-MSCs showed less 6-OHDA induced cell death than control cells in vitro. When Lv-PSPN-MSCs were injected into the striatum of PD rats, we observed the survival rate, migration, differentiation and the behavior change of PD rats. We found that Lv-PSPN-MSCs showed higher survival rate in rat brain compared with Lv-null-MSCs. Rotational behavior showed that rats receiving Lv-PSPN-MSCs showed the most significant improvement compared with those in other groups. HPLC results showed the content of DA in striatum of rats which received Lv-PSPN-MSCs was highest compared with those in other groups. In conclusion, our results suggest that transplantation of Lv-PSPN-MSCs can lead to remarkable therapeutic effects in PD rats.

Glial Cell Line-Derived Neurotrophic Factor Family Members Reduce Microglial Activation via Inhibiting p38MAPKs-Mediated Inflammatory Responses.

Previous studies have shown that glial cell line-derived neurotrophic factor (GDNF) family ligands (GFL) are potent survival factors for dopaminergic neurons and motoneurons with therapeutic potential for Parkinson's disease. However, little is known about direct influences of the GFL on microglia function, which are known to express part of the GDNF receptor system. Using RT-PCR and immunohistochemistrym we investigated the expression of the GDNF family receptor alpha 1 (GFR alpha) and the coreceptor transmembrane receptor tyrosine kinase (RET) in rat microglia in vitro as well as the effect of GFL on the expression of proinflammatory molecules in LPS activated microglia. We could show that GFL are able to regulate microglia functions and suggest that part of the well known neuroprotective action may be related to the suppression of microglial activation. We further elucidated the functional significance and pathophysiological implications of these findings and demonstrate that microglia are target cells of members of the GFL (GDNF and the structurally related neurotrophic factors neurturin (NRTN), artemin (ARTN), and persephin (PSPN)).

Characterization of the nerve-stellate cell interactions in pancreatic cancer

Glial cell line-derived neurotrophic factor (GDNF) is expressed in the gastrointestinal tract of the developing mouse and appears to play an important role in the migration of enteric neuron precursors into and along the small and large intestines. Two other GDNF family members, neurturin and artemin, are also expressed in the developing gut although artemin is only expressed in the esophagus. We examined the effects of GDNF, neurturin, and artemin on neural crest cell migration and neurite outgrowth in explants of mouse esophagus, midgut, and hindgut. Both GDNF and neurturin induced neural crest cell migration and neurite outgrowth in all regions examined. In the esophagus, the effect of GDNF on migration and neurite outgrowth declined with age between E11.5 and E14.5, but neurturin still had a strong neurite outgrowth effect at E14.5. Artemin did not promote neural migration or neurite outgrowth in any region investigated. The effects of GDNF family ligands are mediated by the Ret tyrosine kinase. We examined the density of neurons in the esophagus of Ret−/− mice, which lack neurons in the small and large intestines. The density of esophageal neurons in Ret−/− mice was only about 4% of the density of esophageal neurons in Ret+/− and Ret+/+ mice. These results show that GDNF and neurturin promote migration and neurite outgrowth of crest-derived cells in the esophagus as well as the intestine. Moreover, like intestinal neurons, the development of esophageal neurons is largely Ret-dependent.

Su2031 Down-Regulation of Neurturin in Diverticular Disease -Implications of GDNF Family Ligands (Gfls) for Neuronal Deficits Observed in Diverticular Disease

Su2031 Down-Regulation of Neurturin in Diverticular Disease -Implications of GDNF Family Ligands (Gfls) for Neuronal Deficits Observed in Diverticular Disease

Acute exercise prevents the development of neuropathic pain and the sprouting of non-peptidergic (GDNF- and artemin-responsive) c-fibers after spinal cord injury

Spinal cord injury (SCI) impaired sensory fiber transmission leads to chronic, debilitating neuropathic pain. Sensory afferents are responsive to neurotrophic factors, molecules that are known to promote survival and maintenance of neurons, and regulate sensory neuron transduction of peripheral stimuli. A subset of primary afferent fibers responds only to the glial cell-line derived neurotrophic factor (GDNF) family of ligands (GFLs) and is non-peptidergic. In peripheral nerve injury models, restoration of GDNF or artemin (another GFL) to pre-injury levels within the spinal cord attenuates neuropathic pain. One non-invasive approach to increase the levels of GFLs in the spinal cord is through exercise (Ex), and to date exercise training is the only ameliorative, non-pharmacological treatment for SCI-induced neuropathic pain. The purpose of this study was 3-fold: 1) to determine whether exercise affects the onset of SCI-induced neuropathic pain; 2) to examine the temporal profile of GDNF and artemin in the dorsal root ganglia and spinal cord dorsal horn regions associated with forepaw dermatomes after SCI and Ex; and 3) to characterize GFL-responsive sensory fiber plasticity after SCI and Ex. Adult, female, Sprague–Dawley rats received a moderate, unilateral spinal cord contusion at C5. A subset of rats was exercised (SCI+Ex) on automated running wheels for 20min, 5days/week starting at 5days post-injury (dpi), continuing until 9 or 37dpi. Hargreaves' and von Frey testing was performed preoperatively and weekly post-SCI. Forty-two percent of rats in the unexercised group exhibited tactile allodynia of the forepaws while the other 58% retained normal sensation. The development of SCI-induced neuropathic pain correlated with a marked decrease in the levels of GDNF and artemin in the spinal cord and DRGs. Additionally, a dramatic increase in the density and the distribution throughout the dorsal horn of GFL-responsive afferents was observed in rats with SCI-induced allodynia. Importantly, in SCI rats that received Ex, the incidence of tactile allodynia decreased to 7% (1/17) and there was maintenance of GDNF and artemin at normal levels, with a normal distribution of GFL-responsive fibers. These data suggest that GFLs and/or their downstream effectors may be important modulators of pain fiber plasticity, representing effective targets for anti-allodynic therapeutics. Furthermore, we highlight the potent beneficial effects of acute exercise after SCI.

Immunohistological analysis of neurturin and its receptors in human cochlea

Difficulties in obtaining properly preserved human cochlea have been a major obstacle to in vitro study of this deeply located and hard bone-fortressed hearing organ. Our study aimed at investigating GDNF family ligands (GFLs) and their receptors in the human cochleae that were surgically obtained during a transcochlear approach dealing with life-threatening, intra-cranial meningiomas. The specimens were properly fixed with 4% paraformaldehyde in the operating room. By using immunohistochemical techniques, distribution of GDNF, Neurturin (NTN, one member of GFLs), as well as cRet, GFRα-1 and GFRα-2 receptors in the human cochleae was investigated. Five cochleae from five adult patients were processed for the study. The patients had normal hearing threshold before operation. cRet receptor immunoreactivity was seen in the spiral ganglion neurons, mainly inside the cell bodies but rarely in the nerve fibers and not in the organ of Corti. Immunolabeling for GFRα-1 and GFRα-2 receptors was identified mainly in the cell bodies of the spiral neurons than in the nerve fibers. In the organ of Corti, GFRα-1 immunostaining could be demonstrated in the Deiters' cells, Hensen cells, inner pillar cells, and weakly in the inner hair cells but not in the outer hair cells; no structures in the organ of Corti were labeled with GFRα-2 receptor antibody. NTN immunostaining was found in the supporting cells of organ of Corti, including Deiters' cells, Hensen cells as well as Claudius' cells. In the spiral ganglia, NTN immunostaining was seen in both the cell bodies and the nerve fibers of neurons. GDNF immunoreactivity was not revealed in human cochlea. Surgically obtained human cochleae were properly fixed and underwent immunohistochemical investigation of neurotrophic elements. NTN and its receptors discovered in current study can be responsible for the unique neuronal survival properties in human spiral ganglion (hSG); a prerequisite for the function of cochlear implants.

Site-specific gene expression and localization of growth factor ligand receptors RET, GFRα1 and GFRα2 in human adult colon

Two of the glial-cell-line-derived neurotrophic factor (GDNF) family ligands (GFLs), namely GDNF and neurturin (NRTN), are essential neurotropic factors for enteric nerve cells. Signal transduction is mediated by a receptor complex composed of GDNF family receptor alpha 1 (GFRα1) for GDNF or GFRα2 for NRTN, together with the tyrosine kinase receptor RET (rearranged during transfection). As both factors and their receptors are crucial for enteric neuron survival, we assess the site-specific gene expression of these GFLs and their corresponding receptors in human adult colon. Full-thickness colonic specimens were obtained after partial colectomy for non-obstructing colorectal carcinoma. Samples were processed for immunohistochemistry and co-localization studies. Site-specific gene expression was determined by real-time quantitative polymerase chain reaction in enteric ganglia and in circular and longitudinal muscle harvested by microdissection. Protein expression of the receptors was mainly localized in the myenteric and submucosal plexus. Dual-label immunohistochemistry with PGP 9.5 as a pan-neuronal marker detected immunoreactivity of the receptors in neuronal somata and ganglionic neuropil. RET immunoreactivity co-localized with neuronal GFRα1 and GFRα2 signals. The dominant source of receptor mRNA expression was in myenteric ganglia, whereas both GFLs showed higher expression in smooth muscle layers. The distribution and expression pattern of GDNF and NRTN and their corresponding receptors in the human adult enteric nervous system indicate a role of both GFLs not only in development but also in the maintenance of neurons in adulthood. The data also provide a basis for the assessment of disturbed signaling components of the GDNF and NRTN system in enteric neuropathies underlying disorders of gastrointestinal motility.

GDNF family ligand dependent STAT3 activation is mediated by specific alternatively spliced isoforms of GFRα2 and RET

Neurturin (NRTN), a member of the GDNF family of ligands (GFL), is currently investigated in a series of clinical trials for Parkinson's disease. NRTN signals through its cognate receptor GFRα2 and co-receptor RET to induce neurite outgrowth, but the underlying mechanism remains to be better understood. STAT3 was previously shown to be activated by oncogenic RET, independent of ligand and GFRα. In this study, we demonstrated that NRTN induced serine727 but not tyrosine705 phosphorylation of STAT3 in primary cortical neuron and neuronal cell lines. Remarkably, STAT3 phosphorylation was found to be mediated specifically by GFRα2c and RET9 isoforms. Furthermore, serine but not tyrosine dominant negative mutant of STAT3 impaired NRTN induced neurite outgrowth, indicative of the role of STAT3 as a downstream mediator of NRTN function. Similar to NGF, the NRTN induced P-Ser-STAT3 was localized to the mitochondria but not to the nucleus. Mitochondrial STAT3 was further found to be intimately involved in NRTN induced neurite outgrowth. Collectively, these findings demonstrated the hitherto unrecognized and novel role of specific GFRα2 and RET isoforms in mediating NRTN activation of STAT3 and the transcription independent mechanism whereby the mitochondria localized P-Ser-STAT3 mediated NRTN induced neurite outgrowth.

Characterization of Drosophila GDNF Receptor-Like and Evidence for Its Evolutionarily Conserved Interaction with Neural Cell Adhesion Molecule (NCAM)/FasII

BackgroundGlial cell line-derived neurotrophic factor (GDNF) family ligands are secreted growth factors distantly related to the TGF-β superfamily. In mammals, they bind to the GDNF family receptor α (Gfrα) and signal through the Ret receptor tyrosine kinase. In order to gain insight into the evolution of the Ret-Gfr-Gdnf signaling system, we have cloned and characterized the first invertebrate Gfr-like cDNA (DmGfrl) from Drosophila melanogaster and generated a DmGfrl mutant allele.ResultsWe found that DmGfrl encodes a large GPI-anchored membrane protein with four GFR-like domains. In line with the fact that insects lack GDNF ligands, DmGfrl mediated neither Drosophila Ret phosphorylation nor mammalian RET phosphorylation. In situ hybridization analysis revealed that DmGfrl is expressed in the central and peripheral nervous systems throughout Drosophila development, but, surprisingly, DmGfrl and DmRet expression patterns were largely non-overlapping. We generated a DmGfrl null allele by genomic FLP deletion and found that both DmGfrl null females and males are viable but display fertility defects. The female fertility defect manifested as dorsal appendage malformation, small size and reduced viability of eggs laid by mutant females. In male flies DmGfrl interacted genetically with the Drosophila Ncam (neural cell adhesion molecule) homolog FasII to regulate fertility.ConclusionOur results suggest that Ret and Gfrl did not function as an in cis receptor-coreceptor pair before the emergence of GDNF family ligands, and that the Ncam-Gfr interaction predated the in cis Ret-Gfr interaction in evolution. The fertility defects that we describe in DmGfrl null flies suggest that GDNF receptor-like has an evolutionarily ancient role in regulating male fertility and a previously unrecognized role in regulating oogenesis.SignificanceThese results shed light on the evolutionary aspects of the structure, expression and function of Ret-Gfrα and Ncam-Gfrα signaling complexes.

Gdnf Activates Midline Repulsion by Semaphorin3B via NCAM during Commissural Axon Guidance

The Neurotrophic factor gdnf plays diverse developmental roles, supporting survival and also acting asachemoattractant for axon and cell migration. We report that in the developing spinal cord, a focal source of gdnf is present in the floor plate (FP) where commissural axons cross the midline. Gdnf has nodirect guidance properties but switches on the responsiveness of crossing commissural growth cones to the midline repellent Semaphorin3B by suppressing calpain-mediated processing of the Sema3B signaling coreceptor Plexin-A1. Analysis of single and double mutant mouse models indicates that although gdnf is the principal trigger of Sema3B midline repulsion, it acts with another FP cue, NrCAM. Finally, genetic and invitro experiments provide evidence that this gdnf effect is RET independent and mediated by NCAM/GFRα1 signaling. This study identifies a regulator of midline crossing and reveals interplays between Semaphorin and gdnf signaling during axon guidance.

Regulation of the Na,K-ATPase Gamma-Subunit FXYD2 by Runx1 and Ret Signaling in Normal and Injured Non-Peptidergic Nociceptive Sensory Neurons

Dorsal root ganglia (DRGs) contain the cell bodies of sensory neurons which relay nociceptive, thermoceptive, mechanoceptive and proprioceptive information from peripheral tissues toward the central nervous system. These neurons establish constant communication with their targets which insures correct maturation and functioning of the somato-sensory nervous system. Interfering with this two-way communication leads to cellular, electrophysiological and molecular modifications that can eventually cause neuropathic conditions. In this study we reveal that FXYD2, which encodes the gamma-subunit of the Na,K-ATPase reported so far to be mainly expressed in the kidney, is induced in the mouse DRGs at postnatal stages where it is restricted specifically to the TrkB-expressing mechanoceptive and Ret-positive/IB4-binding non-peptidergic nociceptive neurons. In non-peptidergic nociceptors, we show that the transcription factor Runx1 controls FXYD2 expression during the maturation of the somato-sensory system, partly through regulation of the tyrosine kinase receptor Ret. Moreover, Ret signaling maintains FXYD2 expression in adults as demonstrated by the axotomy-induced down-regulation of the gene that can be reverted by in vivo delivery of GDNF family ligands. Altogether, these results establish FXYD2 as a specific marker of defined sensory neuron subtypes and a new target of the Ret signaling pathway during normal maturation of the non-peptidergic nociceptive neurons and after sciatic nerve injury.

GDNF Family Ligands: A Potential Future for Parkinson's Disease Therapy

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by motor dysfunction that occurs secondary to loss of dopaminergic neurons in the nigrostriatal pathway. Current pharmacotherapies focus on the replacement of lost dopamine to alleviate disease symptoms. However, over time this method of therapy loses effectiveness due to the continued death of dopaminergic neurons. Alternative strategies for the treatment of PD are aimed at modifying the disease state through the preservation of remaining dopamine neurons or even the regeneration of dopamine innervation through the use of neurotrophic factors. Neurotrophic factors are specialized proteins that can promote neuronal development, maintain neuronal health and modulate neuronal function in the ventral midbrain, making them candidates for the treatment of PD. Preclinial studies indicate that members of the glial cell line-derived neurotrophic factor family of ligands are capable of preserving the degenerating dopamine neurons. These promising results moved neurotrophic factor therapy to clinical trials in PD patients. To date, neurotrophic factor therapy is proven to be safe and well-tolerated in humans, but conclusive evidence of efficacy in the clinic remains to be determined. This review will discuss the preclinical and clinical experiments of glial cell line-derived neurotrophic factor family ligands for the treatment of PD.

GDNF family ligand RET receptor in the brain of adult zebrafish

RET is a tyrosine kinase receptor, and transduces signaling by family of glial cell line-derived neurotrophic factor ligands (GFLs). RET is involved in the development of enteric nervous system, of sympathetic, parasympathetic, motor and sensory neurons. RET exists in two main isoforms originated by differential splicing, RET9 and RET51; phylogenetic studies have shown that the RET gene is conserved across vertebrates. The aim of this study was to investigate the RET expression within the brain of zebrafish, using immunohistochemistry, western blotting and RT-PCR. In homogenate brains both RET protein and mRNA were observed. RET immunoreactivity was widespread in neurons and neural processes of all the major regions of the brain. These results demonstrate the occurrence of RET and suggest an involvement of GDNF family ligands in the brain of adult zebrafish.

Neurotrophin and GDNF family ligand receptor expression in vagal sensory nerve subtypes innervating the adult guinea pig respiratory tract

We combined retrograde tracing techniques with single-neuron RT-PCR to compare the expression of neurotrophic factor receptors in nodose vs. jugular vagal sensory neurons. The neurons were further categorized based on location of their terminals (tracheal or lungs) and based on expression of the ionotropic capsaicin receptor TRPV1. Consistent with functional studies, nearly all jugular neurons innervating the trachea and lungs expressed TRPV1. With respect to the neurotrophin receptors, the TRPV1-expressing jugular C-fiber neurons innervating both the trachea and lung compartments preferentially expressed tropomyosin-receptor kinase A (TrkA), with only a minority of neurons expressing TrkB or TrkC. The nodose neurons that express TRPV1 (presumed nodose C-fibers) innervate mainly intrapulmonary structures. These neurons preferentially expressed TrkB, with only a minority expressing TrkA or TrkC. The expression pattern in tracheal TRPV1-negative neurons, nodose tracheal presumed Aδ-fiber neurons as well as the intrapulmonary TRPV1-negative presumed Aβ-fiber neurons, was similar to that observed in the nodose C-fiber neurons. We also evaluated the expression of GFRα receptors and RET (receptors for the GDNF family ligands). Virtually all vagal sensory neurons innervating the respiratory tract expressed RET and GFRα1. The jugular neurons also categorically expressed GFRα3, as well as ∼50% of the nodose neurons. GFRα2 was expressed in ∼50% of the neurons irrespective of subtype. The results reveal that Trk receptor expression in vagal afferent neurons innervating the adult respiratory tract depends more on the location of the cell bodies (jugular vs. nodose ganglion) than either the location of the terminals or the functional phenotype of the nerve. The data also reveal that in addition to neurotrophins, the GDNF family ligands may be important neuromodulators of vagal afferent nerves innervating the adult respiratory tract.

Heparan sulfate proteoglycan syndecan-3 is a novel receptor for GDNF, neurturin, and artemin

Glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) are potent survival factors for dopaminergic neurons and motoneurons with therapeutic potential for Parkinson's disease. Soluble GFLs bind to a ligand-specific glycosylphosphatidylinositol-anchored coreceptor (GDNF family receptor α) and signal through the receptor tyrosine kinase RET. In this paper, we show that all immobilized matrix-bound GFLs, except persephin, use a fundamentally different receptor. They interact with syndecan-3, a transmembrane heparan sulfate (HS) proteoglycan, by binding to its HS chains with high affinity. GFL-syndecan-3 interaction mediates both cell spreading and neurite outgrowth with the involvement of Src kinase activation. GDNF promotes migration of cortical neurons in a syndecan-3-dependent manner, and in agreement, mice lacking syndecan-3 or GDNF have a reduced number of cortical γ-aminobutyric acid-releasing neurons, suggesting a central role for the two molecules in cortical development. Collectively, syndecan-3 may directly transduce GFL signals or serve as a coreceptor, presenting GFLs to the signaling receptor RET.

Ret-Dependent and Ret-Independent Mechanisms of Gfl-Induced Sensitization

BackgroundThe GDNF family ligands (GFLs) are regulators of neurogenic inflammation and pain. We have previously shown that GFLs increase the release of the sensory neuron neuropeptide, calcitonin gene-related peptide (CGRP) from isolated mouse DRG.ResultsInhibitors of the mitogen-activated protein kinase (MAPK) pathway abolished the enhancement of CGRP release by GDNF. Neurturin-induced enhancement in the stimulated release of CGRP, used as an indication of sensory neuronal sensitization, was abolished by inhibition of the phosphatidylinositol-3 kinase (PI-3K) pathway. Reduction in Ret expression abolished the GDNF-induced sensitization, but did not fully inhibit the increase in stimulus-evoked release of CGRP caused by neurturin or artemin, indicating the presence of Ret-independent GFL-induced signaling in sensory neurons. Integrin β-1 and NCAM are involved in a component of Ret-independent GFL signaling in sensory neurons.ConclusionsThese data demonstrate the distinct and variable Ret-dependent and Ret-independent signaling mechanisms by which GFLs induce sensitization of sensory neurons. Additionally, there is a clear disconnect between intracellular signaling pathway activation and changes in sensory neuronal function.

Sciatic nerve injury in adult rats causes distinct changes in the central projections of sensory neurons expressing different glial cell line‐derived neurotrophic factor family receptors

Most small unmyelinated neurons in adult rat dorsal root ganglia (DRG) express one or more of the coreceptors targeted by glial cell line-derived neurotrophic factor (GDNF), neurturin, and artemin (GFRalpha1, GFRalpha2, and GFRalpha3, respectively). The function of these GDNF family ligands (GFLs) is not fully elucidated but recent evidence suggests GFLs could function in sensory neuron regeneration after nerve injury and peripheral nociceptor sensitization. In this study we used immunohistochemistry to determine if the DRG neurons targeted by each GFL change after sciatic nerve injury. We compared complete sciatic nerve transection and the chronic constriction model and found that the pattern of changes incurred by each injury was broadly similar. In lumbar spinal cord there was a widespread increase in neuronal GFRalpha1 immunoreactivity (IR) in the L1-6 dorsal horn. GFRalpha3-IR also increased but in a more restricted area. In contrast, GFRalpha2-IR decreased in patches of superficial dorsal horn and this loss was more extensive after transection injury. No change in calcitonin gene-related peptide-IR was detected after either injury. Analysis of double-immunolabeled L5 DRG sections suggested the main effect of injury on GFRalpha1- and GFRalpha3-IR was to increase expression in both myelinated and unmyelinated neurons. In contrast, no change in basal expression of GFRalpha2-IR was detected in DRG by analysis of fluorescence intensity and there was a small but significant reduction in GFRalpha2-IR neurons. Our results suggest that the DRG neuronal populations targeted by GDNF, neurturin, or artemin and the effect of exogenous GFLs could change significantly after a peripheral nerve injury.