Receptor GFRα1 Research Articles

No neuronal loss, but alterations of the GDNF system in asymptomatic diverticulosis.

BackgroundGlial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor known to promote the survival and maintenance of neurons not only in the developing but also in the adult enteric nervous system. As diverticular disease (DD) is associated with reduced myenteric neurons, alterations of the GDNF system were studied in asymptomatic diverticulosis (diverticulosis) and DD.MethodsMorphometric analysis for quantifying myenteric ganglia and neurons were assessed in colonic full-thickness sections of patients with diverticulosis and controls. Samples of tunica muscularis (TM) and laser-microdissected myenteric ganglia from patients with diverticulosis, DD and controls were analyzed for mRNA expression levels of GDNF, GFRA1, and RET by RT-qPCR. Myenteric protein expression of both receptors was quantified by fluorescence-immunohistochemistry of patients with diverticulosis, DD, and controls.ResultsAlthough no myenteric morphometric alterations were found in patients with diverticulosis, GDNF, GFRA1 and RET mRNA expression was down-regulated in the TM of patients with diverticulosis as well as DD. Furthermore GFRA1 and RET myenteric plexus mRNA expression of patients with diverticulosis and DD was down-regulated, whereas GDNF remained unaltered. Myenteric immunoreactivity of the receptors GFRα1 and RET was decreased in both asymptomatic diverticulosis and DD patients.ConclusionOur data provide evidence for an impaired GDNF system at gene and protein level not only in DD but also during early stages of diverticula formation. Thus, the results strengthen the idea of a disturbed GDNF-responsiveness as contributive factor for a primary enteric neuropathy involved in the pathogenesis and disturbed intestinal motility observed in DD.

Effects of Polymer Encapsulated Glial Cell Line-Derived Neurotrophic Factor Secreting Cells on Odontoblast-like Cell Survival

Objective: Cells that are encapsulated into immunoprotective and implantable devices provide a means of enabling targeted, longterm, continuous, de novo synthesized delivery of very high levels of therapeutic molecules. In this study, we examined effects of polymer encapsulated glial cell line-derived neurotrophic factor (GDNF) secreting ARPE-19 cells on the in vitro viability of odontoblast-like cells (KN-3 cells) under normally lethal starvation conditions. Materials and methods: Devices (polymeric hollow fibers) were loaded with either GDNF-secreting ARPE-19 cells (GDNF-device) or non-modified ARPE-19 cells (ARPE-control device). KN-3 cell survival and function was quantified under starvation conditions when the cells were co-cultured with either control or GDNF devices. We further examined the specificity of GDNF by neutralizing the activity of GDNF with the soluble GDNF receptor (GFRα-1). Results: When KN-3 cells were co-cultured with either device under starvation, the cells exhibited a concentration gradient-dependent enhancement of survival in the area immediately surrounding the device. Similar survival benefits occurred when KN-3 cells were cultured in media previously used to maintain the cells encapsulated in the device. Because ARPE-19 cells also normally secrete low levels of VEGF, the maintenance medium from each device group was pretreated with soluble Flt-1 or soluble GFRα-1 to neutralize VEGF or GDNF. The survival of KN-3 cells was suppressed when cultured in VEGF-neutralized medium from ARPE-control devices, but not in that from GDNF-devices. In contrast, when GDNF was neutralized with soluble GFRα-1, the survival of KN-3 cells was intensely suppressed. Conclusion: These results suggest that GDNF delivered from encapsulated cells can augment the survival of odontoblast-like cells, and that the delivery of growth factors by encapsulated cells may be useful for enhancing pulp wound healing and regeneration. These data also pave the way for additional in vivo studies to determine the utility of this approach in regenerative endodontics.

GFRα1 Regulates Purkinje Cell Migration by Counteracting NCAM Function

SummaryDuring embryonic development of the cerebellum, Purkinje cells (PCs) migrate away from the ventricular zone to form the PC plate. The mechanisms that regulate PC migration are incompletely understood. Here, we report that the neurotrophic receptor GFRα1 is transiently expressed in developing PCs and loss of GFRα1 delays PC migration. Neither GDNF nor RET, the canonical GFRα1 ligand and co-receptor, respectively, contribute to this process. Instead, we found that the neural cell adhesion molecule NCAM is co-expressed and directly interacts with GFRα1 in embryonic PCs. Genetic reduction of NCAM expression enhances wild-type PC migration and restores migration in Gfra1 mutants, indicating that NCAM restricts PC migration in the embryonic cerebellum. In vitro experiments indicated that GFRα1 can function both in cis and trans to counteract NCAM and promote PC migration. Collectively, our studies show that GFRα1 contributes to PC migration by limiting NCAM function.

Age-associated decrease in GDNF and its cognate receptor GFRα-1 protein expression in human skin.

Glial cell line-derived neurotrophic factor (GDNF) and its cognate receptor (GFRα-1) are expressed in normal human skin. They are involved in murine hair follicle morphogenesis and cycling control. We hypothesize that 'GDNF and GFRα-1 protein expression in human skin undergoes age-associated alterations. To test our hypothesis, the expression of these proteins was examined in human skin specimens obtained from 30 healthy individuals representing three age groups: children (5-18years), adults (19-60years) and the elderly (61-81years). Immunofluorescent and light microscopic immunohistologic analyses were performed using tyramide signal amplification and avidin-biotin complex staining methods respectively. GDNF mRNA expression was examined by RT-PCR analysis. GDNF mRNA and protein as well as GFRα-1 protein expressions were detected in normal human skin. We found significantly reduced epidermal expression of these proteins with ageing. In the epidermis, the expression was strong in the skin of children and declined gradually with ageing, being moderate in adults and weak in the elderly. In children and adults, the expression of both GDNF and GFRα-1 proteins was strongest in the stratum basale and decreased gradually towards the surface layers where it was completely absent in the stratum corneum. In the elderly, GDNF and GFRα-1 protein expression was confined to the stratum basale. In the dermis, both GDNF and GFRα-1 proteins had strong expressions in the fibroblasts, sweat glands, sebaceous glands, hair follicles and blood vessels regardless of the age. Thus there is a decrease in epidermal GDNF and GFRα-1 protein expression in normal human skin with ageing. Our findings suggest that the consequences of this is that GFRα-1-mediated signalling is altered during the ageing process. The clinical and therapeutic ramifications of these observations mandate further investigations.

114. Replication-Defective HSV Vector Development for Targeted Gene Delivery

Gene therapy treatment strategies for Huntington's disease (HD) depend on a vector that (i) allows targeted delivery of large therapeutic transgenes to specific cell populations and (ii) is replication-defective and non-cytotoxic. Thus, our goal is to develop a replication-defective HSV-based gene therapy vector to deliver therapeutic transgenes to the cells most affected by expression of the mutant Huntington gene (Htt). Strategies for full retargeting of HSV require virus detargeting from its cognate receptors (HVEM and nectin1), recognized by the virus attachment/entry component glycoprotein D (gD), and introduction of a new ligand into gD that allows entry through recognition of the corresponding cellular receptors. To target an HSV vector for entry exclusively into cells expressing the receptor GFRα1 we employed the ligand GDNF. We replaced the signal peptide and HVEM binding domain of gD with pre-pro-(pp)GDNF to create a GFRα1 targeting protein, gD(Y38)_GDNF, that can still bind nectin1. Virus expressing gD(Y38)_GDNF was propagated on cells expressing nectin1 and purified virus was shown to enter nectin1-deficient J1.1-2 and B78H1 cells in a GFRα1-dependent manner. U2OS cells engineered to express GFRα1 demonstrated the most robust virus entry and spread, allowing us to create and propagate a fully retargeted virus that can no longer bind nectin1, gD(Δ38)_GDNF. Once the functionality of gD(Δ38)_GDNF was confirmed, we transferred this mutation to a replication-defective backbone lacking essential immediate early gene expression. Complementing cells were screened and those cells that best supported growth of the replication-defective backbone were selected. Virus stocks were propagated and tested for receptor specificity in vitro and experiments are currently underway to assess GFRα1-specific entry in vivo. We anticipate that the use of neuronal-specific targeting ligands, such as GFRα1, in replication-defective backbones will promote homing of non-toxic HSV gene therapy vectors to cells affected in HD.

Neurotrophic factors: promising candidates in tissue regeneration.

Neurotrophic factors: promising candidates in tissue regeneration.

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

The formation of synaptic connections during nervous system development requires the precise control of dendrite growth and synapse formation. Although glial cell line-derived neurotrophic factor (GDNF) and its receptor GFRα1 are expressed in the forebrain, the role of this system in the hippocampus remains unclear. Here, we investigated the consequences of GFRα1 deficiency for the development of hippocampal connections. Analysis of conditional Gfra1 knockout mice shows a reduction in dendritic length and complexity, as well as a decrease in postsynaptic density specializations and in the synaptic localization of postsynaptic proteins in hippocampal neurons. Gain- and loss-of-function assays demonstrate that the GDNF-GFRα1 complex promotes dendritic growth and postsynaptic differentiation in cultured hippocampal neurons. Finally, in vitro assays revealed that GDNF-GFRα1-induced dendrite growth and spine formation are mediated by NCAM signaling. Taken together, our results indicate that the GDNF-GFRα1 complex is essential for proper hippocampal circuit development.

Immunohistochemical Analysis of GDNF and Its Cognate Receptor GFRα-1 Protein Expression in Vitiliginous Skin Lesions.

Vitiligo is an idiopathic skin disease, characterized by circumscribed white macules or patches on the skin due to loss of the functional melanocytes. Glial cell line-derived neurotrophic factor (GDNF) and its cognate receptor (GFRα-1) are distal members of the transforming growth factor-β superfamily. GDNF, produced by the basal cell keratinocytes, is involved in the migration and differentiation of the melanocytes from the neural crest to the epidermis. This study examines the hypothesis that expression of GDNF protein and its cognate receptor GFRα-1 protein is altered in vitiliginous skin. To test our hypothesis, we examined the expression patterns of these proteins in vitiliginous and corresponding healthy (control) skin biopsies (20 specimens each) using immunoperoxidase staining techniques. We found variations between the vitiliginous skin and healthy skin. In healthy skin, the expression of GDNF and GFRα-1 proteins was strong (basal cell keratinocytes and melanocytes), moderate (spinous layer), and weak (granular cell layer). In contrast, weak expression of GDNF protein was observed in all epidermal layers of vitiliginous skin. GFRα-1 protein expression was strong (basal cell keratinocytes and melanocytes), moderate (spinous layer), and weak (granular cell layer). In both healthy skin and vitiliginous skin, the expression of GDNF and GFRα-1 proteins was strong in the adnexal structures. We report, for the first time, decreased expression of GDNF proteins in the epidermal keratinocytes of vitiliginous skin. Our findings suggest possible pathogenetic roles for these proteins in the development of vitiligo. The clinical ramifications of these observations mandate further investigations.

Glial cell line-derived neurotrophic factor induces cell proliferation in the mouse urogenital sinus.

Glial cell line-derived neurotrophic factor (GDNF) is a TGFβ family member, and GDNF signals through a glycosyl-phosphatidylinositol-linked cell surface receptor (GFRα1) and RET receptor tyrosine kinase. GDNF signaling plays crucial roles in urogenital processes, ranging from cell fate decisions in germline progenitors to ureteric bud outgrowth and renal branching morphogenesis. Gene ablation studies in mice have revealed essential roles for GDNF signaling in urogenital development, although its role in prostate development is unclear. We investigated the functional role of GDNF signaling in the urogenital sinus (UGS) and the developing prostate of mice. GDNF, GFRα1, and RET show time-specific and cell-specific expression during prostate development in vivo. In the UGS, GDNF and GFRα1 are expressed in the urethral mesenchyme (UrM) and epithelium (UrE), whereas RET is restricted to the UrM. In each lobe of the developing prostate, GDNF and GFRα1 expression declines in the epithelium and becomes restricted to the stroma. Using a well-established organ culture system, we determined that exogenous GDNF increases proliferation of UrM and UrE cells, altering UGS morphology. With regard to mechanism, GDNF signaling in the UrM increased RET expression and phosphorylation of ERK1/2. Furthermore, inhibition of RET kinase activity or ERK kinases suppressed GDNF-induced proliferation of UrM cells but not UrE cells. We therefore propose that GDNF signaling in the UGS increases proliferation of UrM and UrE cells by different mechanisms, which are distinguished by the role of RET receptor tyrosine kinase and ERK kinase signaling, thus implicating GDNF signaling in prostate development and growth.

Abstract 4999: Paracrine regulation of glioma invasion by astrocytes is mediated by glial derived neurothropic factor

Abstract Purpose: Glial cells have been shown to play a role in glioma progression. Here we investigate the mechanism by which astrocytes which are abundant in glioma tumors, promote cancer cell invasion. Experimental Design: In this study, we evaluated the effects of astrocytes on glioma biology both in vitro and in vivo and determined the downstream paracrine effect of glial derived neurotropic factor (GDNF) on tumor invasion. Results: Astrocytes conditioned media (ACM) significantly increased human and murine glioma cells migration compared to controls. This effect was inhibited when the activity of GDNF on glioma cells was blocked by RET-Fc chimera or anti-GDNF Ab and by siRNA directed against GDNF expression by astrocytes. Glioma cell incubation with ACM led to time dependent phosphorylation of the GDNF receptor, RET and downstream AKT phosphorylation. Tumor migration and GDNF-RET-AKT activation was inhibited by the RET small molecule inhibitor pyrazolopyrimidine-1 and by the AKT inhibitor LY294002. Finally, blocking of RET by pyrazolopyrimidine-1 or knockout of the RET co receptor GFRα1 in glioma cells reduced the size of brain tumors in immunocompetent mice. Conclusions: We suggest a mechanism by which astrocytes attracted to the glioma microenvironment facilitate brain invasion by secretion of GDNF and activation of RET/GFRα1 receptors expressed by the cancer cells. Note: This abstract was not presented at the meeting. Citation Format: Moran Amit, Ayelet Shabtay-Orbach, Yoav Binenbaum, Ziv Gil. Paracrine regulation of glioma invasion by astrocytes is mediated by glial derived neurothropic factor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4999. doi:10.1158/1538-7445.AM2014-4999

Deficiency of GDNF Receptor GFRα1 in Alzheimer's Neurons Results in Neuronal Death.

We have recently developed aged cortical neuron cultures from autopsied human brains with Alzheimer's disease (AD). During the culturing process, we found that glutamatergic cortical neurons from the AD brain lacked a response to glial cell line-derived neurotrophic factor (GDNF), including no axonal regrowth, and were starting to undergo apoptosis. Here we showed that, in cortical neurons from age- and gender-matched cognitively normal control (NC) subjects (NC neurons), GDNF enhanced the expression of GDNF family receptor subtype α1 (GFRα1), but not the other three subtypes (GFRα2, GFRα3, and GFRα4), whereas GDNF failed to induce GFRα1 expression in cortical neurons from the AD brain (AD neurons). The exogenous introduction of GFRα1, but not of its binding partner α1-neural cell adhesion molecule, or RET into AD neurons restored the effect of GDNF on neuronal survival. Moreover, between NC and AD neurons, the AMPA receptor blocker CNQX and the NMDA receptor blocker AP-5 had opposite effects on the GFRα1 expression induced by GDNF. In NC neurons, the presence of glutamate receptors was necessary for GDNF-linked GFRα1 expression, while in AD neurons the absence of glutamate receptors was required for GFRα1 expression by GDNF stimulation. These results suggest that, in AD neurons, specific impairments of GFRα1, which may be linked to glutamatergic neurotransmission, shed light on developing potential therapeutic strategies for AD by upregulation of GFRα1 expression.

Expression of neurotrophic factors and their receptors in the carotid body of spontaneously hypertensive rats

The carotid body (CB) is a small neural crest-derived structure that senses oxygen levels in blood and monitors ventilation. The spontaneously hypertensive rat (SHR) is considered a good experimental model for primary hypertension and is extensively used to study cardiovascular diseases. The hypertensive CB shows structural plasticity and could enlarge without vasodilation. Our immunohistochemical studies revealed the presence of nuclear Ki-67 protein in the sustentacular cells, nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3 and their corresponding receptors p75NTR, TrkA, TrkB and TrC in the majority of glomus cells and also in a subset of sustentacular cells. In addition, virtually all glomus cells expressed glial cell line-derived neurotrophic factor and its specific receptor GFRα1. The present study demonstrates that in glomus cells of hypertensive animals there is enhanced expression of components of the neurotrophin signaling system compared to normotensive rats. Our results suggest that the elevated production of neurotrophic factors in SHRs could explain CB and sympathetic hyperactivity leading to hypertension.

Brn4 and TH synergistically promote the differentiation of neural stem cells into dopaminergic neurons

Neural stem cells (NSCs) are pluripotent cells capable of differentiation into dopaminergic (DA) neurons, which are the major cell types damaged in Parkinson's disease (PD). Therefore, NSCs are considered the most promising cell source for cell replacement therapy of PD. However, the poor differentiation and maturation of DA neurons and decreased cell survival after transplantation are a challenge. We have previously demonstrated that Brn4, a member of the POU domain family of transcription factors, induced the differentiation of NSCs into neurons and promoted their maturation. In this study, we directly transduced tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis, into NSCs to induce DA neuronal differentiation. However, these DA neurons were morphologically immature and seldom expressed dopamine transporter (DAT), a late marker of mature DA neurons. In contrast, TH co-transfected with Brn4 generated increased number of mature DA neurons. Furthermore, Brn4 significantly induced the expression of glial cell line-derived neurotrophic factor (GDNF) with its receptors GFRα-1 and Ret, which may contribute to the maturation and survival of differentiated DA neurons. Our findings may be of future importance for the use of NSCs in cell replacement therapy of PD.

Hypoxia-inducible factor 1α regulates branching morphogenesis during kidney development

The kidneys are exposed to hypoxic conditions during development. Hypoxia-inducible factor (HIF), an important mediator of the response to hypoxia, is believed to have an important role in development. However, the relationship between HIF and branching morphogenesis has not been elucidated clearly.In this study, we examined whether HIF regulates kidney development. We harvested kidneys from day 13 rat embryos (E13Ks) and cultured the organs under normoxic (20% O2/5% CO2) or hypoxic (5% O2/5% CO2) conditions. We evaluated the kidneys based on morphology and gene expression. E13Ks cultured under hypoxic conditions had significantly more ureteric bud (UB) branching than the E13Ks cultured under normoxic conditions. In addition, the mRNA levels of GDNF and GDNF receptor (GFR-α1), increased under hypoxic conditions in E13Ks. When we cultured E13Ks with the HIF-1α inhibitor digoxin or with siRNA targeting HIF-1α under hypoxic conditions, we did not observe increased UB branching. In addition, the expression of GDNF and GFR-α1 was inhibited under hypoxic conditions when the kidneys were treated with siRNA targeting HIF-1α. We also elucidated that hypoxia inhibited UB cell apoptosis and promoted the expression of FGF7 mRNA levels in metanephric mesenchymal (MM) cells in vitro.These findings suggest that hypoxic condition has important roles in inducing branching morphogenesis during kidney development. Hypoxia might mediate branching morphogenesis via not only GDNF/Ret but also FGF signaling pathway.

The role of SorLA in neurotrophic activity and complex behavior

SorLA binds amyloid precursor protein (APP) in the trans-Golgi network and protects it against proteolysis by both the alpha- and beta-secretases, preventing the generation of toxic amyloid-beta peptide. We have recently found that SorLA also affects neuronal function in a different manner by acting as sorting receptor for the neurotrophic factor GDNF and its receptors GFRα1 and RET, directing them from the cell surface to endosomes. Through this mechanism, GDNF is targeted to lysosomes and degraded while GFRα1 and RET recycles, creating an efficient GDNF clearance pathway. SorLA thereby influences a number of neuronal systems known to depend on GDNF activity including the dopaminergic system and the hippocampus. Accordingly, mice with altered SorLA expression exhibit altered reward and memory function, and a behavioral phenotype characterized by altered anxiety, hyperactivity, and response to psychostimulants.

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.

Glial cell line-derived neurotrophic factor (GDNF) induced migration of spermatogonial cells in vitro via MEK and NF-kB pathways

Glial cell line-derived neurotrophic factor (GDNF) regulates spermatogonial stem cell (SSC) maintenance. In the present study, we examined the levels and the cellular origin of GDNF in mouse testes during age-development, and the capacity of GDNF to induce migration of enriched GFR-α1 positive cells in vitro. The involvement of MAP kinase (MEK) and NF-kB signal pathways were examined. Our results show high levels of GDNF in testicular tissue of one-week-old mice which significantly decreased with age when examined by ELISA, real time PCR (qPCR) and immunofluorescence staining (IF) analysis. GDNF receptor (GFR-α1) expression was similar to GDNF when examined by qPCR analysis. Only Sertoli cell cultures (SCs) from one-week-old mice produced GDNF compared to SCs from older mice. However, peritubular cells from all the examined ages did not produce GDNF. The addition of recombinant GDNF (rGDNF) or supernatant from SCs from one-week-old mice to GFR-α1 positive cells induced their migration in vitro. This effect was significantly reduced by the addition of inhibitors to MEK (PD98059, U0126), NF-kB (PDTC) and IkB protease inhibitor (TPCK).Our results show for the first time the capacity of rGDNF and supernatant from SCs to induce migration of enriched GFR-α1 positive cells, and the possible involvement of MEK, NF-kB and IkB in this process. This study may suggest a novel role for GDNF in the regulation SSC niches and spermatogenesis.

The GDNF System Is Altered in Diverticular Disease – Implications for Pathogenesis

Background & AimsAbsence of glial cell line-derived neurotrophic factor (GDNF) leads to intestinal aganglionosis. We recently demonstrated that patients with diverticular disease (DD) exhibit hypoganglionosis suggesting neurotrophic factor deprivation. Thus, we screened mRNA expression pattern of the GDNF system in DD and examined the effects of GDNF on cultured enteric neurons.MethodsColonic specimens obtained from patients with DD (n = 21) and controls (n = 20) were assessed for mRNA expression levels of the GDNF system (GDNF, GDNF receptors GFRα1 and RET). To identify the tissue source of GDNF and its receptors, laser-microdissected (LMD) samples of human myenteric ganglia and intestinal muscle layers were analyzed separately by qPCR. Furthermore, the effects of GDNF treatment on cultured enteric neurons (receptor expression, neuronal differentiation and plasticity) were monitored.ResultsmRNA expression of GDNF and its receptors was significantly down-regulated in the muscularis propria of patients with DD. LMD samples revealed high expression of GDNF in circular and longitudinal muscle layers, whereas GDNF receptors were also expressed in myenteric ganglia. GDNF treatment of cultured enteric neurons increased mRNA expression of its receptors and promoted neuronal differentiation and plasticity revealed by synaptophysin mRNA and protein expression.ConclusionsOur results suggest that the GDNF system is compromised in DD. In vitro studies demonstrate that GDNF enhances expression of its receptors and promotes enteric neuronal differentiation and plasticity. Since patients with DD exhibit hypoganglionosis, we propose that the observed enteric neuronal loss in DD may be due to lacking neurotrophic support mediated by the GDNF system.

SorLA Controls Neurotrophic Activity by Sorting of GDNF and Its Receptors GFRα1 and RET

Glial cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor that has reached clinical trials for Parkinson's disease. GDNF binds to its coreceptor GFRα1 and signals through the transmembrane receptor tyrosine kinase RET, or RET independently through NCAM or syndecan-3. Whereas the GDNF signaling cascades are well described, cellular turnover and trafficking of GDNF and its receptors remain poorly characterized. Here, we find that SorLA acts as sorting receptor for the GDNF/GFRα1 complex, directing it from the cell surface to endosomes. Through this mechanism, GDNF is targeted to lysosomes and degraded while GFRα1 recycles, creating an efficient GDNF clearance pathway. The SorLA/GFRα1 complex further targets RET for endocytosis but not for degradation, affecting GDNF-induced neurotrophic activities. SorLA-deficient mice display elevated GDNF levels, altered dopaminergic function, marked hyperactivity, and reduced anxiety, all of which are phenotypes related to abnormal GDNF activity. Taken together, these findings establish SorLA as a critical regulator of GDNF activity in the CNS.

Critical Role of GFRα1 in the Development and Function of the Main Olfactory System

Glial cell line-derived neurotrophic factor (GDNF) and its receptor GFRα1 are prominently expressed in the olfactory epithelium (OE) and olfactory bulb (OB), but their importance for olfactory system development is completely unknown. We have investigated the consequences of GFRα1 deficiency for mouse olfactory system development and function. In the OE, GFRα1 was expressed in basal precursors, immature olfactory sensory neurons (OSNs), and olfactory ensheathing cells (OECs), but was excluded from mature OSNs. The OE of newborn Gfra1 knock-out mice was thinner and contained fewer OSNs, but more dividing precursors, suggesting deficient neurogenesis. Immature OSN axon bundles were enlarged and associated OECs increased, indicating impaired migration of OECs and OSN axons. In the OB, GFRα1 was expressed in immature OSN axons and OECs of the nerve layer, as well as mitral and tufted cells, but was excluded from GABAergic interneurons. In newborn knock-outs, the nerve layer was dramatically reduced, exhibiting fewer axons and OECs. Bulbs were smaller and presented fewer and disorganized glomeruli and a significant reduction in mitral cells. Numbers of tyrosine hydroxylase-, calbindin-, and calretinin-expressing interneurons were also reduced in newborn mice lacking Gfra1. At birth, the OE and OB of Gdnf knock-out mice displayed comparable phenotypes. Similar deficits were also found in adult heterozygous Gfra1(+/-) mutants, which in addition displayed diminished responses in behavioral tests of olfactory function. We conclude that GFRα1 is critical for the development and function of the main olfactory system, contributing to the development and allocation of all major classes of neurons and glial cells.