Molecular Vision 2004; 10:289-296 <http://www.molvis.org/molvis/v10/a37/> Received 12 February 2004 | Accepted 31 March 2004 | Published 19 April 2004

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Effect of exogenous neurotrophins on Trk receptor phosphorylation, cell proliferation, and neurotrophin secretion by cells isolated from the human lamina cribrosa

Wendi S. Lambert,¹ Abbot F. Clark,^1,2 Robert J. Wordinger¹
 
 

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: Dr. Wendi S. Lambert, Department of Medicine, Duke University Medical Center, DUMC 3445, Genome Science Research Building 1, 595 LaSalle Street, Durham, NC, 27710; Phone: (919) 684-0701; FAX: (919) 684-0938; email: wlambert@chg.duhs.duke.edu

Abstract

Purpose: Glaucoma is the number one cause of preventable blindness in the United States. The lamina cribrosa (LC) region of the optic nerve head (ONH) is a major site of injury in glaucomatous optic neuropathy. Neurotrophins (NTs), which include NGF, BDNF, NT-3, and NT-4, are growth factors involved in the development and support of neurons and in non-neuronal interactions. Cells within the human LC express high affinity tyrosine kinase receptors (Trks) for NTs. The purpose of this study was to determine if exogenous NTs cause (a) phosphorylation of Trk receptors in LC cells and ONH astrocytes and (b) cell proliferation and/or secretion of NTs by LC cells and ONH astrocytes.

Methods: Trk phosphorylation in response to exogenous NGF, BDNF, NT-3, and NT-4 treatment was studied in LC cells and ONH astrocytes using immunoprecipitation and Western blotting. Cell number was assayed following treatment with exogenous NTs or the Trk phosphorylation inhibitor compound K-252a. Secretion of NTs following exogenous administration of NTs was determined using immunoassays.

Results: LC cells and ONH astrocytes express Trk receptors that are phosphorylated in response to exogenous NTs. Autocrine/paracrine signaling was also evident by Trk phosphorylation in the absence of exogenous NT treatment. ONH astrocyte cell number increased following exogenous treatment with each NT. LC cell number increased following exogenous NGF or NT-3 treatment only. Treatment with the Trk phosphorylation inhibitor K-252a decreased both LC and ONH astrocyte cell number. Exogenous NT treatment increased the secretion of NGF by LC cells and ONH astrocytes. BDNF secretion by LC cells and ONH astrocytes was decreased by exogenous NT treatment.

Conclusions: LC cells and ONH astrocytes express functional Trk receptors that can be activated in response to exogenous NTs. The activation of Trk receptors expressed by LC cells and ONH astrocytes in the absence of exogenous NT treatment suggests autocrine/paracrine NT signaling may occur within the ONH. Neurotrophin signaling in LC cells and ONH astrocytes may regulate cell number and/or NT secretion within the LC region of the ONH.

Introduction

Glaucoma is the number one cause of preventable blindness in the United States, affecting an estimated 3 million Americans (Glaucoma Research Foundation). Glaucoma is an optic neuropathy defined by characteristic optic nerve head (ONH) and associated visual field changes due to the gradual death of retinal ganglion cells. A unique feature of glaucoma is the deeply excavated appearance of the ONH [1] due to the progressive posterior displacement and compression of the lamina cribrosa (LC) and its disinsertion from the sclera rim [2]. The LC region of the ONH consists of connective tissue plates aligned to form channels that guide and support retinal ganglion cell (RGC) axons as they exit the eye to form the optic nerve [3,4]. Two major cell types, ONH astrocytes and LC cells, have been isolated from the human LC [2,5]. ONH astrocytes express glial fibrillary acidic protein (GFAP) and separate the nerve fibers from the connective tissue plates in the LC [3]. Lamina cribrosa cells are characterized by a lack of GFAP immunoreactivity and are believed to reside within or between the connective tissue plates [2,3,5].

Neurotrophins (NTs) are polypeptide growth factors that promote neuronal development, differentiation, and survival. Included in this family is nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). In addition to being trophic factors for neuronal cells, NTs also induce the expression and/or secretion of other NTs [6,7]. A variety of cellular responses in non-neuronal cells are also regulated by NTs. For example, NTs promote the survival of endothelial cells, fibroblasts, and retinal pigmented epithelium [8-10]. Proliferation in response to NTs has been observed in brain astrocytes, Müller cells, cardiac myocytes, and microglia [11-14]. Other responses to NTs include changes in morphology and differentiation [10,15].

Neurotrophins signal through two types of receptors, Trk receptors and p75. Trk receptors are tyrosine kinase receptors that homodimerize and autophosphorylate upon ligand binding [16]. Trk A specifically binds NGF, Trk B binds BDNF and NT-4, and Trk C binds NT-3. Complicating this system are truncated Trk B and C receptors that lack the tyrosine kinase domain. No function for the truncated receptors has been determined, although it is believed these receptors may inhibit NT signaling by blocking Trk autophosphorylation [17]. The extracellular domains of the truncated Trks are identical to the full-length receptors, which would allow for ligand binding but not autophosphorylation. The p75 NT receptor can bind all four NTs. In certain cells, p75 expression is required for Trk A to effectively bind NGF [18].

In a previous study, we showed NT and Trk receptor expression in human ONH tissue [19]. In addition, we demonstrated that cultured human LC cells and ONH astrocytes express and secrete NTs, and also express Trk receptors [19]. The expression of NTs and full-length Trk receptors by LC cells and ONH astrocytes suggests the possibility of autocrine/paracrine NT signaling within the LC. However, these cells also express truncated Trk receptors and do not express p75 at detectable levels [19]. In order to determine if LC cells and ONH astrocytes were capable of responding to NTs, we examined the phosphorylation of Trk receptors following exogenous NT treatment. Similarly, we examined several cellular responses following exogenous NT treatment in our cells. The purpose of this study was to determine if cells from the human LC express functional Trk receptors, and what responses NT signaling in these cells may regulate.

Methods

Materials

DMEM and fetal bovine serum (FBS) were purchased from HyClone Labs, Logan, UT. The following materials were purchased from Sigma-Aldrich, St. Louis, MO; 0.25% trypsin solution, DMSO, protein A-agarose beads, and human recombinant NGF, BDNF, NT-3 and NT-4. The following materials were purchased from Gibco BRL Life Technologies, Grand Island, NY; L-glutamine, penicillin/streptomycin and fungizone (amphotericin B). Costar 96-well plates and Nunc ELISA/EIA 96 well Maxisorp plates were purchased from Fisher Scientific, Pittsburgh, PA. Polyclonal antibodies to Trk A, Trk B, Trk C, truncated Trk B (Trk B.T) and phosphorylated Trk were purchased from Santa Cruz Biotechnology Inc, Santa Cruz, CA. CellTiter 96® Aq_ueous Non-Radioactive Cell Proliferation Assays and Emax® ImmunoAssay Systems specific for NGF, BDNF, and NT-3 were purchased from Promega Corporation, Madison, WI.

Cell culture

Lamina cribrosa and ONH astrocyte cell lines were obtained from human LC explants from separate donors as described previously [19]. Cells were cultured in Ham's F-10 Media (LC cells; JRH Biosciences, Lenexa, KS) or DMEM (astrocytes) supplemented with 10% FBS, L-glutamine (0.292 mg/ml), penicillin (100 units/ml)/streptomycin (0.1 mg/ml), and amphotericin B (4 μg/ml). Cells were passaged using a 0.25% trypsin solution. All cultures were maintained in 5% CO₂/95% air at 37 °C and media were changed every 2 to 3 days. Characterization of these cells was performed as described previously [2,5,19]. Cells expressing α-smooth muscle actin that did not express glial fibrillary acidic protein (GFAP) were characterized as LC cells [2,19]. Cells expressing GFAP and neural cell adhesion molecule (N-CAM) were characterized as optic nerve head (ONH) astrocytes [2,19,20]. Both cell types expressed extracellular matrix proteins, such as collagen I, collagen III, collagen IV and elastin [2,5,19]. Adult cell lines from donors whose ages ranged from 39 years to 90 years were used in the following experiments.

Immunoprecipitation, SDS-PAGE and western blotting of activated Trk receptors

One adult LC and one adult ONH astrocyte cell line were treated with serum free media for 24 h prior to treatment with 50 ng/ml exogenous NT in serum free media. To block Trk phosphorylation [21] cells were pre-treated with 500 ng/ml K-252a, an inhibitor of Trk phosphorylation (resuspended in DMSO; ICN Biomedicals Inc., Aurora, OH), for 3 h and then treated with NT in serum free media for 10 min. Cellular protein was collected in lysis buffer modified from Watson et al. [22] (20 mM Tris (pH 7.4), 137 mM NaCl, 1% NP40, 10% glycerol, 48 mM sodium fluoride, 16 mM sodium pyrophosphate, 1 mM PMSF, 20 μM leupeptin, 10 μg/ml aprotinin, and 1 mM sodium orthovanadate (10 μl/ml)). Protein concentration was measured using the Bio-Rad D_c Protein Assay System (Bio-Rad Laboratories, Richmond, CA). Cellular lysate (75 μg) was incubated overnight with 1.5 μg anti-Trk A, Trk B, Trk C or Trk B.T antibody. Samples were then incubated with protein A-agarose for 2 h at 4 °C, washed 4 times, and resuspended in sample buffer. Immunoprecipitated proteins were separated on 7.5% denaturing polyacrylamide gels and electrophoretically transferred to nitrocellulose membranes. Blots were processed using a goat anti-phospho-Trk antibody, a donkey anti-goat IgG HRP-labeled secondary antibody (Santa Cruz Biotechnology Inc.), and the ECL Western detection system (Amersham, Arlington Heights, IL). Blots were exposed to Hyperfilm-ECL (Amersham) for various times depending on the amount of target protein present.

Capture antibody specificity was determined by immunoprecipitating LC cell lysate (300 μg) with 6 μg anti-Trk A, Trk B, Trk C, or Trk B.T antibody. Following incubation with protein A-agarose, samples were separated by SDS-PAGE and transferred to nitrocellulose. Four identical blots were processed using the appropriate anti-Trk antibody, secondary antibody and ECL Western detection system. Control immunoprecipitations (IPs) using ONH astrocyte cell lysate were also performed and included the following: IP in the absence of cell lysate, IP in the absence of capture antibody (anti-Trk B), and IP with an irrelevant capture antibody (rabbit anti-glucocorticoid receptor, Affinity Bioreagent Inc., Golden, CO).

Determination of cell number following exogenous neurotrophin treatment

Adult LC cells and ONH astrocytes were trypsinized, counted using a hemacytometer and plated into Costar 96-well plates at a density of 1,000 cells/well. Cells were allowed to attach overnight and were then placed in serum free media for 24 h. Cell number was assayed using the CellTiter 96® Aq_ueous Non-Radioactive Cell Proliferation Assay following treatment with human recombinant NGF, BDNF, NT-3 or NT-4 (50 ng/ml) in Ham's F-10 or DMEM containing 0.5% FBS for 7 days. On day 4 of the treatment, cell morphology was examined, media was replaced, and fresh NT was added. On day 7, media was removed and replaced with 100 μl of serum free media. Twenty microliters of MTS/PMS solution was added to each well. Plates were incubated at 5% CO₂/95% air at 37°C for 1 h, at which time the absorbance at 490 nm was read using a SpectraMax® 190 microplate reader and Softmax® Pro (Molecular Devices Corporation, Sunnyvale, CA). Metabolically active cells convert MTS into formazan, which is soluble in aqueous solutions. The quantity of the formazan product measured by the amount of absorbance at 490 nm is therefore directly proportional to the number of living cells. Cell number per well was calculated from a standard curve generated using known amounts of cells per well. A standard curve was generated for each cell line assayed. Three LC cell lines and three ONH astrocyte cell lines were assayed. The entire experiment, including standard curves, was repeated twice. Changes in cell number following NT treatment were reported as a percent of the 0.5% FBS control.

Determination of cell number following treatment with K-252a, an inhibitor of Trk phosphorylation

LC cells and ONH astrocytes were plated as described above. Following a 24 h treatment with serum free media, cells were treated with 500 ng/ml K-252a or 500 ng/ml DMSO (vehicle) in Ham's F-10 (LC cells) or DMEM (ONH astrocytes) containing 0.5% FBS for 24 h. Cell number was assayed as described above. Three LC cell lines and three ONH astrocyte cell lines were assayed. The entire experiment, including standard curves, was repeated twice. Changes in cell number following K-252a or DMSO treatment were reported as a percent of the 0.5% FBS control.

Immunoassay of conditioned media following exogenous neurotrophin treatment

Twenty-four hours prior to exogenous NT treatment, pre-confluent LC cells and ONH astrocytes were placed in serum free media. Human recombinant NGF, BDNF, NT-3 or NT-4 (50 ng/ml) in media containing 0.5% FBS was used to treat LC cells and ONH astrocytes for 48 h. Following treatment, conditioned media was collected and concentrated using Millipore Centriplus YM-3 Centrifugal Filter Devices (Millipore Corporation, Bedford, MA). Emax® ImmunoAssay Systems specific for NGF, BDNF, NT-3, and NT-4 were performed according to manufacturer's instructions. Conditioned media was added to Nunc ELISA/EIA 96 well Maxisorp plates coated with anti-NT polyclonal antibodies. Secreted NT was detected by treating the plates with the respective NT monoclonal 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 NT standard included in each assay was used to generate a standard curve that was used to calculate the amount of secreted NT per well. The amount of secreted NT per sample was normalized to total protein per sample. Samples were assayed in triplicate. Conditioned media from three LC cell lines and three ONH astrocyte cell lines were assayed. Each immunoassay was repeated twice. Changes in NT secretion following NT treatment were reported as a percent of the 0.5% FBS control.

Results

Activation of Trk receptors in LC cells and ONH astrocytes following exogenous NT treatment

Figure 1 represents phosphorylated Trk A and Trk C expression following exogenous NGF or NT-3 treatment of LC cells and ONH astrocytes. Treatment with NGF resulted in the phosphorylation of Trk A in LC cells and ONH astrocytes (lanes 1 and 5). The phospho-Trk antibody detected two bands, which could represent the two isoforms of Trk A [16]. A lower level of Trk A phosphorylation was observed in LC cells treated with NGF plus K-252a, a Trk phosphorylation inhibitor (lane 2). Trk A phosphorylation in ONH astrocytes treated with NGF plus K-252a was similar to that of NGF treatment alone (lane 6). Addition of K-252a in the absence of exogenous NGF reduced Trk A phosphorylation in LC cells and ONH astrocytes (lanes 3 and 7). Trk A phosphorylation was also detected in untreated LC cells and ONH astrocytes, which served as controls (lanes 4 and 8). Trk A activation appeared to be higher in untreated ONH astrocytes than in ONH astrocytes treated with exogenous NGF, or exogenous NGF plus K-252a. It is possible that Trk receptors on ONH astrocytes were saturated with endogenous NGF and the addition of exogenous NGF could not increase Trk A phosphorylation.

Treatment of LC cells and ONH astrocytes with NT-3 produced multiple phosphorylated Trk C bands in LC cells and ONH astrocytes. At least four full length Trk C isoforms have been identified [16] which could explain the multiple bands detected by the phospho-Trk antibody. Both cell types demonstrated Trk C phosphorylation in response to exogenous NT-3 or NT-3 plus K-252a (lanes 1, 2, 5 and 6). Low levels of phosphorylated Trk C were observed in untreated LC cells and ONH astrocytes (lanes 4 and 8), and treatment with K-252a alone decreased Trk C phosphorylation in both cell types (lanes 3 and 7).

Activation of Trk B receptors following exogenous NT treatment

The phosphorylation of Trk B in response to exogenous BDNF and NT-4 is shown in Figure 2. Treatment with BDNF or NT-4 increased Trk B phosphorylation in LC cells and ONH astrocytes (lanes 1 and 5). This phosphorylation was decreased by the addition of K-252a, both in the presence and absence of exogenous NT (lanes 2, 3, 6 and 7). Trk B phosphorylation was also observed in untreated LC cells and ONH astrocytes (lanes 4 and 8), although slight variations in Trk B phosphorylation were observed in untreated ONH astrocytes. Multiple untreated flasks from the same ONH astrocyte cell line were collected, and this difference could be explained by variations among individual cultures at the time of collection. This experiment was repeated using an antibody to Trk B.T for the immunoprecipitations. The extracellular domain of Trk B.T is identical to Trk B, but Trk B.T lacks the tyrosine kinase domain and therefore would not be phosphorylated. No Trk phosphorylation was observed in LC cells or ONH astrocytes treated with NT, NT plus K-252a, or in the controls (data not shown). Lysate that did not undergo immunoprecipitation with the Trk B.T antibody was included as a positive control for phosphorylation.

Appropriate controls for the immunoprecipitations were performed in order to determine if the capture antibodies used to immunoprecipitate a specific Trk receptor would non-specifically bind other Trk receptors. Anti-Trk A antibody immunoprecipitated Trk A and none of the other Trk receptor proteins. Similar results were observed for the anti-Trk B, anti-Trk C and anti-Trk B.T antibodies (data not shown). As a control for the immunoprecipitation protocol, Trk B was immunoprecipitated from ONH astrocyte cell lysate, various components were omitted, and samples were processed using the anti-phospho-Trk antibody. No phospho-Trk B was detected in samples when the cell lysate or capture antibody was omitted, or when an irrelevant capture antibody was used (data not shown). Phospho-Trk expression was detected in samples when all components were present and in samples that did not undergo immunoprecipitation. No Trk phosphorylation was detected when primary antibody (anti-phospho-Trk) was omitted (data not shown).

Exogenous neurotrophin treatment increases cell number

We have shown previously that exogenous NGF treatment results in proliferation of LC cells [19]. In order to expand on this observation, LC and ONH astrocyte cell number was determined following treatment with exogenous NTs for 7 days (Figure 3). Treatment of LC cells with exogenous NGF or NT-3 resulted in a 13-17% increase in cell number, which was statistically significant when compared to the 0.5% FBS control. Although there was a trend for BDNF and NT-4 treatment to increase LC cell number, this increase was not statistically significant. A statistically significant increase in ONH astrocyte cell number (22-33%) was observed following treatment with exogenous NGF, BDNF, NT-3 or NT-4 when compared to the 0.5% FBS control. Lamina cribrosa cell and ONH astrocyte morphology was examined on day four of the seven day NT treatment (data not shown). Treatment of LC cells and ONH astrocytes with any of the exogenous NTs did not alter morphology compared to cells cultured in 0.5% FBS-media. Cells cultured in 10% FBS-media were similar in morphology to LC cells and ONH astrocytes described previously [2,5,19].

Treatment with K-252a decreases LC and ONH astrocyte cell number

To confirm that NT signaling results in LC and ONH astrocyte cell proliferation, cell number was measured following treatment with K-252a, a Trk phosphorylation inhibitor (Figure 4). The addition of K-252a resulted in a statistically significant decrease (30%) in LC cell number. A similar decrease (33%) was observed in ONH astrocytes treated with K-252a. The addition of vehicle alone (DMSO) did not significantly affect LC or ONH astrocyte cell number indicating the change in cell number was due to the inhibition of Trk phosphorylation by K-252a and not due to toxicity. The decrease in cell number following K-252a treatment did not appear to be due to cell death, as cell morphology was similar to the controls (data not shown).

Exogenous neurotrophin treatment increases NGF secretion and decreases BDNF secretion

In other cell types, NTs have been shown to induce the expression and/or secretion of other NTs. We examined NT secretion by LC cells and ONH astrocytes following exogenous NT treatment (Figure 5). Treatment of LC cells and ONH astrocytes with BDNF, NT-3 or NT-4 resulted in a statistically significant increase in NGF secretion. Treatment with NT-4 increased NGF secretion by LC cells almost 270%, while ONH astrocyte NGF secretion increased nearly 190% in response to BDNF or NT-3. In contrast, a statistically significant decrease in BDNF secretion was observed by LC cells and ONH astrocytes in response to NGF, NT-3 or NT-4 treatment. Following treatment with NT-4, BDNF secretion was decreased to 16% of the control in LC cells and 24% of the control in ONH astrocytes. We were unable to detect the secretion of NT-3 or NT-4 by LC cells or ONH astrocytes following NT treatment.

Discussion

We have previously demonstrated that cells from the human LC express NTs and Trk receptors and are capable of secreting NTs [19]. In this study we examined Trk receptor activation in human LC cells and ONH astrocytes following exogenous NT treatment. The results demonstrated that these cell types express functional Trk receptors that can be phosphorylated in response to exogenous NTs. Trk receptors expressed by LC cells and ONH astrocytes were also phosphorylated in the absence of exogenous NT, suggesting autocrine or paracrine signaling via endogenous NT sources occurs in LC cells and ONH astrocytes. Interestingly, activation of Trk receptors on LC cells and ONH astrocytes via exogenous NTs resulted in physiological responses including modest cell proliferation and the regulation of NGF and BDNF secretion.

The expression of NTs and Trk receptors by LC cells and ONH astrocytes along with NT secretion suggested these cells were capable of autocrine/paracrine NT signaling [19]. However, efficient binding of NGF to Trk A may be compromised [18] in LC cells and ONH astrocytes due to the absence of p75 expression in these cells [19]. Cells from the LC also express truncated Trk receptors, which may hinder NT signaling by inhibiting Trk phosphorylation [23-26]. We observed Trk A receptor phosphorylation in LC cells and ONH astrocytes following treatment with NGF suggesting that p75 expression is not required by LC cells and ONH astrocytes to activate Trk A. Phosphorylation of Trk B and Trk C receptors following exogenous BDNF, NT-4 and NT-3 treatment was observed in LC cells and ONH astrocytes indicating that NT signaling through full-length Trk B or Trk C can occur in cells from the LC in the presence of truncated Trk B and Trk C isoforms. Lamina cribrosa cells and ONH astrocytes that were not treated with exogenous NT also demonstrated low levels of Trk receptor phosphorylation suggesting autocrine/paracrine NT signaling occurs within these cells. Neurotrophin secreted by a cell within the LC could potentially bind and activate self-expressed Trk receptors (autocrine signaling) or Trk receptors on adjacent cells (paracrine signaling). To our knowledge, this is the first time autocrine/paracrine NT signaling has been shown in cells isolated from the human LC.

LC cells and ONH astrocytes could utilize autocrine/paracrine NT signaling to regulate the microenvironment of the LC. Neurotrophin signaling has been shown to influence a variety of responses in non-neuronal cell types including survival, proliferation, differentiation, morphologic changes, and gene expression [10,13,27,28]. A study by Sörby et al. [29] suggests a balance between proliferation and growth inhibition is regulated by the level of receptor tyrosine phosphorylation within a cell. Since Trk receptors are tyrosine kinase receptors, variations in Trk phosphorylation in LC cells and ONH astrocytes could influence cell proliferation. A modest increase (13-33%) in cell number was observed following a 7 day treatment with a single exogenous NT. Cell number was significantly decreased (30-33%) when cells were treated for 24 h with K-252a, a compound that inhibits phosphorylation at all three full-length Trk receptors. All or most autocrine/paracrine NT signaling through Trk A, Trk B and Trk C was blocked with this treatment, which would explain why a greater decrease in cell number was observed with the shorter K-252a treatment, when compared to the exogenous NT treatment. A larger increase in cell number may have been observed had cells been treated with exogenous NT every day for 7 days, or with a combination of exogenous NTs. ONH astrocyte and LC cell proliferation were affected by both increases (treatment with NT) and decreases (treatment with K-252a) in Trk phosphorylation, indicating autocrine/paracrine NT signaling modulates cell number within the ONH.

A second cellular response to NT signaling we examined in LC cells and ONH astrocytes was NT secretion. In certain neuronal cells, NT secretion can be induced by treatment with exogenous NTs [6,7]. Exogenous NT treatment increased the secretion of NGF by LC cells and ONH astrocytes, suggesting NT signaling could induce NGF secretion within the LC. In contrast to NGF secretion, BDNF secretion was decreased following treatment with exogenous NTs. This could imply that BDNF secretion is negatively regulated by NT signaling within the LC, and also suggests that the various NTs have distinct roles within the LC. The inability to detect NT-3 or NT-4 secretion by LC cells and ONH astrocytes in this study could be due to autocrine/paracrine signaling. Secreted NT-3 that immediately bound Trk C receptors expressed by LC cells and ONH astrocytes would not be detected in the media. The same is true of secreted NT-4 that bound Trk B or Trk B.T receptors. The detection of NGF and BDNF in conditioned media would imply that LC cells and ONH astrocytes secrete more of these NTs than self expressed Trk receptors are able to bind.

In conclusion, we have shown that LC cells and ONH astrocytes express Trk receptors that can be activated in response to NT binding. In addition, we have demonstrated that autocrine/paracrine NT signaling occurs within LC cells and ONH astrocytes, suggesting it may also occur within the LC region of the human ONH. Autocrine/paracrine NT signaling between LC cells and ONH astrocytes may regulate cell number and/or NT secretion within the LC. For example, BDNF secreted within the LC could activate Trk B receptors on LC cells and/or ONH astrocytes, which could in turn increase the secretion of NGF from neighboring cells (Figure 6). This secreted NGF would activate Trk A receptors expressed by these cells, resulting in decreased secretion of BDNF within the LC. This situation could be important in regulating the microenvironment of the LC, possibly affecting cell number or other cellular responses to NT signaling within LC cells and ONH astrocytes. Exploring signaling molecules downstream of Trk receptors could determine other responses to NT signaling within LC cells and ONH astrocytes. Further studies examining NT signaling in LC cells and ONH astrocytes from glaucomatous donors or in an in vivo model of glaucoma could determine the role of NTs in the progression of glaucoma. Together, these studies would provide a better understanding of glaucoma pathogenesis, which hopefully lead to new therapeutic strategies.

Acknowledgements

The authors would like to acknowledge and thank The Glaucoma Foundation for their generous support, The Central Florida Lions Eye and Tissue Bank for ocular tissue, and Sherry English-Wright, Dr. William Howe, and Paula Billman for their technical assistance. Research underlying this article was made possible by Alcon Laboratories and National Institutes of Health EY12783.

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