Molecular Vision 2003; 9:249-256 <http://www.molvis.org/molvis/v9/a36/> Received 19 February 2003 | Accepted 10 June 2003 | Published 16 June 2003

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Cells of the human optic nerve head express glial cell line-derived neurotrophic factor (GDNF) and the GDNF receptor complex

Robert J. Wordinger,¹ Wendi Lambert,¹ Rajnee Agarwal,¹ Xiaochun Liu,¹ Abbot F. Clark^1,2
 
 

¹Department of Cell Biology and Genetics, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX; ²Glaucoma Research, Alcon Research Ltd., Fort Worth, TX

Correspondence to: Robert J. Wordinger, Ph.D. Department of Cell Biology and Genetics, University of North Texas Health Science Center at Fort Worth, 3500 Camp Bowie Boulevard, Fort Worth, TX; Phone: (817) 735-2045; FAX: (817) 735-2610; email: rwording@hsc.unt.edu

Abstract

Purpose: Glial cell-line derived neurotrophic factor (GDNF) is a distant member of the TGFβ family of growth factors and has wide ranging effects within the central nervous system. In the present study we profile the expression of GDNF and its receptor complex (Ret and GFRα-1) in cells isolated from the human optic nerve head (ONH).

Methods: Lamina cribrosa (LC) cells and ONH astrocytes were used from normal donors of various ages. Total RNA was isolated and subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) to examine mRNA expression of GDNF, Ret, and GFRα-1. Western immunoblotting and immunohistochemistry was used to study protein expression of GDNF and GDNF receptor complex proteins in cultured ONH cells. An immunoassay system (ELISA) was used to examine secretion of GDNF by ONH cells. Cell proliferation was examined following exogenous administration of GDNF.

Results: Lamina cribrosa cells, ONH astrocytes, and LC tissues expressed messenger RNA for GDNF, Ret and GFRα-1. Lamina cribrosa cells and ONH astrocytes also expressed protein for GDNF, Ret, and GFRα-1. Secretion of GDNF by either cell type was not detected. Exogenous GDNF caused a significant increase in cell proliferation of LC cells but not ONH astrocytes.

Conclusions: Cells from the human lamina cribrosa express mRNA and protein for GDNF and its receptor complex. LC cells proliferate in response to exogenous GDNF. The potential for autocrine and/or paracrine GDNF signaling thus exists within the lamina cribrosa, a tissue involved in glaucoma pathogenesis.

Introduction

The term glaucoma is used to describe a group of optic neuropathies characterized by optic nerve damage and visual field loss and often associated with elevated intraocular pressure. An estimated 67 million people worldwide have glaucoma, including 3 million in the United States [1]. In primary open angle glaucoma (POAG) there is a compression or excavation of the optic nerve head (ONH), especially at the level of the lamina cribrosa (LC). The LC is composed of glial cell columns and connective tissue plates that align to form channels that guide and support ganglion cell axons as they exit the eye [2]. The exact mechanism underlying ganglion cell injury in POAG is still unknown.

Glial derived neurotrophic factor (GDNF) is the founding member of a subgroup of peptide trophic factors that are distant members of the transforming growth factor beta (TGFβ) superfamily [3-5]. Other members of the GDNF-family ligands (GFLs) include neurturin, artemin, and persephin [6]. Members of the GFL family are cysteine-knot proteins and function as homodimers. GDNF was first discovered by Lin et. al. [7] and was shown to be a trophic factor for dopaminergic neurons. Subsequently, GDNF has been shown to promote the survival of numerous neuronal populations [8-12].

In addition to its actions within the nervous system, recent evidence suggests GDNF may regulate non-neuronal interactions. For example, GDNF expression within the testis is believed to regulate spermatogonial cell differentiation [13,14]. In addition, tooth development may be dependent on GDNF signaling [15]. In both cases, the expression of GDNF did not correlate with innervation, suggesting a non-neuronal function for GDNF within these tissues. GDNF has been shown to be essential for kidney formation by promoting ureteric budding and branching [16,17]. This effect is possibly due to the ability of GDNF to act as a chemoattractant for kidney epithelial cells, causing them to migrate toward a localized source of GDNF [18].

The effects of GDNF are mediated via a receptor complex consisting of a tyrosine kinase receptor (Ret) and a GDNF binding protein (GFRα-1). Ret, a proto-oncogene isolated by Takahashi et. al. [19], was recognized as a receptor although its ligand was unknown until the discovery of GDNF. Mice lacking functional Ret and GDNF knockout mice demonstrated similar phenotypes [16,17,20,21], suggesting an association between the ligand and receptor. Further study revealed GDNF could also bind a receptor-like protein (GFRα-1) attached to the plasma membrane via a glycoslyphosphotidyl-inositol (GPI)-linkage [22-24]. This localizes the complex to lipid rafts associated with the outer leaflet of the plasma membrane. In all cell types studied thus far, the expression of GFRα-1 is necessary for GDNF induced Ret activation [24,25]. Although Ret activation requires GFRα-1, there is evidence GDNF can also signal via GFRα-1 in the absence of Ret [26]. Ret independent GDNF signaling has been shown in cochlear neurons, all cortical layers, the lateral geniculate nucleus and in the superior colliculus [27,28].

Recent evidence suggests GDNF could be a potential neuroprotective factor for retinal ganglion cells (RGC) since it has been reported to promote the survival of axotomized RGC [29,30]. Within the LC, two major cell populations have been identified: (a) optic nerve head (ONH) astrocytes and (b) lamina cribrosa (LC) cells [31-33]. GDNF may serve a non-neuronal function within the LC and be involved in maintaining the normal microenvironment. This later function would require signaling between LC cells and/or ONH astrocytes. It is unknown at this time whether LC cells and ONH astrocytes express GDNF and the GDNF receptor complex. The objective of this study was to determine the expression of GDNF, Ret and GFRα-1 by cells isolated from the human LC. In addition, we determined if LC cells and ONH astrocytes secrete GDNF and if these cells can respond to exogenous GDNF.

Methods

Cell culture

Human LC explants were obtained and cultured as described previously [33,34]. Briefly, human donor eyes were received from regional eye banks within 24 h of death. The LC was dissected away from the remaining ocular tissue, cut into 3 to 4 explants and placed in culture plates containing DMEM (HyClone Labs, Logan, UT) plus 10% fetal bovine serum (FBS, HyClone Labs) [31]. Lamina cribrosa cells that grew out of the explants were cultured in Ham's F-10 Media (JRH Biosciences, Lenexa, KS) with 10% FBS (HyClone). ONH astrocytes were isolated from populations as previously described [33,34]. Cultured ONH astrocytes were maintained in DMEM plus 10% FBS. All cells were passaged using a 0.25% trypsin solution and were maintained in 5% CO₂/95% O₂ at 37 °C. Human optic nerve head astrocytes and lamina cribrosa cells were generated from donors aged 2 days to 90 years.

RT-PCR

Total cellular RNA was prepared using the TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA). First strand cDNA synthesis and details of the PCR procedure used in our laboratory have been published previously [35]. Primers for GDNF, Ret, and GFRα-1 were designed using Oligo 5.0 software (National Biosciences, Plymouth, MN). The primer pairs, expected product sizes, and annealing temperatures are listed in Table 1. All primer pairs were designed so that amplification of potentially contaminated genomic DNA sequences would produce mRNA PCR products that would be substantially larger than expected, because intron sequences that were excised during RNA processing would be included in genomic DNA. The β-actin PCR primers, 5' AGG CCA ACC GCG AGA AGA TGA CC 3' (upstream) and 5' GAA GTC CAG GGC GAC GTA GCA C 3' (downstream) with an annealing temperature of 55 °C yielded a PCR product of 350 base pairs upon RT-PCR and gel electrophoresis. To ensure specificity of the RT-PCR products, Southern blot analysis was performed (data not shown) with probes designed using Primer 3 [36] that hybridized to a region within the amplified PCR product.

Immunohistochemistry

Cells were grown on glass coverslips, fixed with 3.5% (v/v) formaldehyde and then treated with 0.2% (v/v) Triton X-100 (Fisher Scientific, Pittsburgh, PA). Non-specific binding was blocked by a 20 min incubation with 10% (v/v) normal serum (Gibco BRL Life Technologies). Primary antibodies were purchased from Santa Cruz Biotechnology Inc., Santa Cruz, CA. Coverslips were incubated with primary antibodies (1:100 in 1.5% (v/v) normal serum) for 1 h at room temperature, followed by a 45 min incubation in appropriate Alexa Fluor^TM 488-labeled secondary antibodies (1:200 in 1.5% (v/v) normal serum; Molecular Probes, Inc., Eugene, OR). Coverslips incubated in 1.5% (v/v) normal serum in place of primary antibody served as negative controls. Coverslips were then mounted on clean glass slides using Aquamount (Lerner Laboratories Inc., Pittsburgh, PA). Images were captured using a Nikon Microphot FXA scope (Nikon, Inc., Melville, NY) equipped with an FITC filter and a SenSys CCD camera (Photometrics, Tucson, AZ). Images were deconvolved using a Macintosh Power Mac G3 (Macintosh, Cupertino, CA) and Scanalytics IPLAB (Scanalytics, Fairfax, VA) and Vaytek Microtome (Vaytek, Fairfield, IA) software.

Western immunoblotting

Total cellular protein was extracted from cultured cells as described previously [33]. Protein concentration was measured using the Bio-Rad Dc Protein Assay System (Bio-Rad Laboratories, Richmond, CA). The cellular lysate was separated on denaturing polyacrylamide gels and then transferred by electrophoresis to nitrocellulose membranes. Blots were incubated with primary antibodies (See immunohistochemistry), processed with horseradish peroxidase conjugated secondary antibodies, and proteins were visualized using ECL detection reagents (Amersham, Arlington Heights, IL). Blots were then exposed to Hyperfilm-ECL (Amersham) for various times depending on the amount of target protein present.

Immunoassay for secreted GDNF

Conditioned media was collected from pre-confluent lamina cribrosa and ONH astrocytes after a 72 h treatment with serum-free media containing 0.5 mg/ml BSA. The media was concentrated using Centriplus centrifugal filter devices (Millipore Corporation, Bedford, MA). The GDNF Emax^TM ImmunoAssay System (Promega, Madison WI) specific for GDNF was performed according to manufacturer's instructions. Media was added to Nunc ELISA/EIA 96 well Maxisorp plates (Fisher Scientific) coated with anti-GDNF monoclonal antibodies. Secreted GDNF was detected by treating the plates with a GDNF polyclonal antibody followed by a horseradish peroxidase conjugated secondary antibody. Enzyme substrate was added to generate a color product whose absorbance was read at 450 nm. A GDNF standard was included in each assay and was used to generate a standard curve.

Cell proliferation assay

Nearly confluent LC cells and ONH astrocytes were trypsinized and seeded in triplicate into 6 well plates at a density of 10,000 cells per well in 2 ml media. Cells were allowed to attach for 24 h and were then treated with serum-free media. Cells were washed 12 h later twice with serum-free media and treated with either 50 ng/ml human recombinant GDNF (R & D Systems Inc, Minneapolis, MN) in serum free media, serum free media alone, or with media containing 10% FBS. Cells were maintained in 5% CO₂/95% O₂ at 37 °C and the media was changed every 2 days during the 14 day treatment. Cells were then washed twice with serum-free media, trypsinized and counted using a Coulter Counter ZM (Beckman Coulter, Inc., Chaska, MN).

Results

Expression of GDNF and GDNF receptor complex mRNA in human ONH cells and tissues

Human optic nerve head astrocytes and lamina cribrosa cells were generated from donors aged 2 days to 90 years. Amplification products of expected size for GDNF, Ret, GFRα-1, and β-actin in adult cell lines the LC are shown in Figure 1. All LC and ONH astrocyte cell lines expressed message for GDNF and GFRα-1. Ret mRNA was observed in all cell lines with the exception of the 66 year old LC sample. Several samples demonstrated multiple bands possibly indicating the presence of Ret isoforms. All cDNA samples from cultured cell lines and tissue samples underwent PCR amplification of β-actin in order to ensure that no genomic DNA contamination was present. The β-actin primer pair was designed to span exons so that the amplification of contaminating genomic DNA sequences would produce a PCR product substantially larger (790 bp) than the expected mRNA product (350 bp). Normal brain astrocyte (NBA) mRNA was utilized for a positive control sample. Control reactions lacking cDNA did not result in amplification products (lanes C) indicating that reagents and primers were free of DNA or RNA contamination. Southern blot analysis demonstrated that the designed primers were specific for GDNF, Ret, and GFRα-1 (data not shown).

Expression of GDNF and GDNF receptor complex proteins in human ONH cells

Representative immunofluorescent staining for GDNF and GDNF receptor complex proteins in adult LC cells and ONH astrocytes are shown in Figure 2 and Figure 3 respectively. Both LC cells and ONH astrocytes demonstrated positive staining for GDNF, Ret, and GFRα-1. No significant differences in intensity or staining pattern were observed between the two cell types. Immunoreactivity for GDNF appeared cytoplasmic with intense staining concentrated around the nucleus. A punctate staining pattern was observed for Ret and GFRα-1 in both cell types. No specific staining was observed when primary antibody was omitted.

Figure 4 represents chemiluminescent detection of GDNF ligand and its receptor proteins in adult cell lines from the ONH. The expression of GDNF was observed in two adult LC cells lines and two adult ONH astrocyte cell lines at molecular weights of 160, 92 and 62 kDa. Cultured LC cells and ONH astrocytes also expressed the GDNF receptor complex proteins Ret and GFRα-1 (molecular weights ranging from 34 kDa to 235 kDa). The expression of β-actin (45 kDa) was used as a positive control.

Immunoassay for GDNF in Conditioned Media of ONH Cells

The secretion of GDNF by cultured cells from the human ONH as detected using an immunoassay (ELISA). The range of GDNF detection in the immunoassay is 16-1000 pg/ml. We were not able to detect GDNF in serumless conditioned media collected from either LC cells or ONH astrocytes. A normal standard curve was obtained indicating that the immunoassay was functional (data not shown). The minimum detectable dose (MDD) was determined by adding two standard deviations to the mean optical density for 20 standard replicates and calculating the corresponding concentration. The MDD for this assay was 30 pg/ml.

Promotion of ONH cell proliferation by exogenous GDNF

Proliferation on ONH cells in response to exogenous GDNF is shown in Table 2. LC cells treated with human recombinant GDNF resulted in an increase in proliferation of 93.4% compared with serum-free controls. Proliferation was also observed in ONH astrocytes following GDNF treatment, however this increase (7.3%) was not significant when compared to serum free controls. Neither cell type proliferated as well in serumless media with GDNF as compared to growth media containing serum (Ham's F-10 or DMEM plus 10% FBS).

Discussion

This study examined the expression of GDNF and the GDNF receptor complex by cells isolated from the human ONH. We have demonstrated that LC cells and ONH astrocytes express mRNA and protein for GDNF, Ret, and GFRα-1. To our knowledge, this is the first time that the expression of GDNF and members of the GDNF receptor complex have been shown in cells isolated from the human ONH. In addition we demonstrate that LC cells proliferate in response to exogenous GDNF.

Lamina cribrosa cells and ONH astrocytes have been characterized previously [31-33]. Both cell types are known to produce various extracellular matrix proteins and are believed to be involved in maintaining the normal microenvironment of the LC [31,32,37-39]. In addition, a re-activation of astrocytes accompanied by gliosis has also been observed in the glaucomatous ONH [40-43]. Since our laboratory has previously demonstrated that ONH cells express and secrete neurotrophins [33], we were interested to determine if other potential neuroprotective factors were also expressed. By determining which factors are expressed by ONH cells, we will be in a better position to understand the local changes that may occur during POAG.

Our RT-PCR results demonstrate that LC cells and ONH astrocytes express mRNA for GDNF, Ret and GFRα-1. Alternative splicing of the GDNF transcript results in two mRNA isoforms, but because the deletion occurs in the prepro region, both transcripts result in identical mature proteins [10,44]. In designing the primer pair for GDNF, we focused on the mRNA sequence downstream from the prepro region of the transcript, therefore our primers produced only one amplification product. Message for GDNF and its receptor complex were translated into protein within cultured LC cells and ONH astrocytes as demonstrated by positive immunofluorescent staining and Western blots. We detected GDNF protein expression in cultured LC cells and ONH astrocytes at 62, 92 and 160 kDa. Protein dimers of GDNF have a reported molecular weight of 33-45 kDa [45]. Jing et al. [24] reported GDNF/GFRα-1 complexes at molecular weights of 75 kDa and 150 kDa. GDNF bound to Ret (170 kDa) has a molecular weight of 185 kDa [24]. Because our cells express protein for GDNF, GFRα-1 and Ret, it is likely that various combinations of these proteins will be present. In addition GDNF is processed from a larger molecular weight precursor form, which would be detected by the antibody utilized in this study.

We were unable to detect GDNF secretion by cultured LC cells or ONH astrocytes. It is possible that our cells did indeed secrete GDNF, but at levels that were below the sensitivity range of the immunoassay (minimum detection of 30 pg/ml). GDNF signaling in cultured LC cells and/or ONH astrocytes could take place between adjacent cells (paracrine) where high concentrations of GDNF are not needed. This would explain the lack of detectable secreted GDNF in conditioned media in this study. Also because the cells express the GDNF receptor complex, secreted GDNF may be attached to receptor complexes to signal in an autocrine fashion. Thus small amounts of soluble GDNF would not be detected. However, our results do indicate that LC cells are expressing a functional GDNF receptor complex since exogenous GDNF caused a significant increase in cell proliferation.

Determining a potential function for GDNF within the ONH is complicated. The LC is considered part of the CNS, however message for GDNF has generally been reported to be low in cells from the adult CNS [46]. GDNF mRNA has been detected in tissues innervated by sympathetic neurons [10], however the ONH does not receive sympathetic innervation. Therefore, it is unlikely that GDNF expression within the LC carries out a function such as target derived neurotrophic support. Message for GDNF has been detected during development in mesenchyme and in mesenchyme derived tissues [47]. The LC is formed from scleral and choroidal tissue, both of which develop from mesoderm and invading neural crest cells [48]. The expression of GDNF mRNA by cells in the LC could therefore be the result of a general mesenchymal expression pattern and neural crest origin.

Retinal ganglion cells have been shown to express GDNF receptors and to respond to GDNF after injury [29,30]. GDNF and GFRα-1 were shown to be upregulated in neurons and astrocytes after injury or inflammation [28-30,49-55]. Ischemia has been suggested as a possible mechanism of injury in POAG. Ischemia results in cellular damage due to a variety of secondary changes, such as increased glutamate, calcium influx, and the generation of nitric oxide and reactive oxygen intermediates. There is evidence that GDNF protects cells after ischemic injury. For example, an increase in GDNF and GFRα-1 mRNA in adult rat brain was observed after global forebrain ischemia [56]. GDNF was able to protect neurons from ischemia, in part by blocking the increase in nitric oxide that accompanies middle cerebral artery occlusion [49]. The promoter region of GDNF could provide a possible explanation for this protection against ischemic injury. The GDNF promoter contains three metal response elements (MREs), which upregulate the transcription of GDNF in response to elevated levels of certain metals (i.e. cadmium, zinc and copper) [57,58]. GDNF transcription via MREs can also be activated by oxidative stress [59]. Oxidative stress is known to activate metal-response transcription factor-1 binding activity [57]. The investigation of GDNF in LC cells and ONH astrocytes under ischemic conditions is currently underway.

At the present time it is unknown if RGC express GDNF receptors along their axons, especially within the LC. ONH astrocytes surround the retinal ganglion cell axon bundles and separate them from the extracellular matrix. It is possible that ONH astrocytes could provide sufficient GDNF for RGC through a juxtacrine/paracrine mechanism. Further studies localizing Ret and/or GFRα-1 to axons within the human LC will be necessary to determine if ONH cells could provide paracrine GDNF support for RGC.

An alternative role for GDNF within the LC is also probable. It is possible that GDNF does not act directly on RGC axons but rather acts in a non-neuronal function within the human ONH. This study demonstrated the co-localization of GDNF and its receptors in LC cells and ONH astrocytes. Non-neuronal GDNF signaling has been observed in numerous tissues. As indicated previously, the normal development of the kidney requires the expression of GDNF in kidney mesenchyme and GFRα-1 and Ret expression in the uteric bud [60]. Mice null for GDNF or Ret show complete renal agenesis, suggesting non-neuronal GDNF signaling is crucial within the kidney [16,17]. Non-neuronal GDNF expression has been shown to regulate spermatogonial cell fate and proliferation of Sertoli cells during early postnatal rat testis development [13,14]. Signaling between GDNF expressing astrocytes and GFRα-1 expressing endothelial cells may be responsible for the maintenance of the blood brain barrier within the cortex [61]. The migration of MDCK cells and pancreatic cancer cells toward a GDNF source implies GDNF may act as a chemoattractant for non-neuronal cells [18,62].

Thus, within the human LC, GDNF could regulate the proliferation and/or differentiation of LC cells and ONH astrocytes, as well as astrocyte-endothelial cell junctions. It is also possible that since LC cells and ONH astrocytes produce extracellular matrix proteins within the LC, GDNF signaling may serve to regulate the expression of these proteins. Lastly, within the ONH, GDNF may be sequestered within the extracellular matrix (ECM) since it has been demonstrated that GDNF is capable of binding heparan sulfate side-chains within proteoglycans [5]. Thus even the release of small quantities of GDNF (e.g. not detectable by ELISA assay) may prove physiologically important in the human ONH since attachment to the ECM would restrict diffusion and raise local concentrations.

An additional important consideration for GDNF expression and function is the report that GDNF is not active unless supplemented by TGFβ[63]. GDNF appears to need co-signaling through TGFβby a mechanism reported to involve stabilization and recruitment of GFRα by TFGβ. The expression of TGFβ isoforms and their receptors have been observed within the LC [64,65], thus suggesting that GDNF within the LC would be active and able to signal. Lastly, a signaling pathway between Ret and NGF via the Trk a receptor has also been demonstrated [66]. While Ret phosphorylation was reported to be independent of GDNF and GFRα-1, it did required activation of the Trk A receptor via NGF. We have previously demonstrated that cells of the human ONH express neurotrophins and Trk receptors and are capable of secreting NGF [33]. Thus within the human ONH the additional possibility exists that Ret signaling occurs independent of GDNF or other GFL family members.

In conclusion, the results of this study indicate that LC cells and ONH astrocytes within the human LC express GDNF and its receptors. The function of GDNF signaling within the LC is unknown at this time. Further studies demonstrating the cellular processes regulated by GDNF signaling in LC cells and ONH astrocytes are needed. The expression of GDNF, Ret and GFRα-1 within the LC under ischemic conditions may provide a better understanding of some of the cellular changes that may occur in POAG.

Acknowledgements

Research underlying this article was made possible by the Morgan Stanley & Co., Inc. Research Fund of the Glaucoma Foundation (New York, NY), Alcon Research Ltd., (Fort Worth, TX.) and the National Institutes of Health (Bethesda, MD; R01 EY12783 to RJW). The authors would like to acknowledge and thank The Central Florida Lions Eye and Tissue Bank for ocular tissue, and Sherry English-Wright for her technical assistance.

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