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Originally published In Press as doi:10.1074/jbc.M402872200 on July 23, 2004

J. Biol. Chem., Vol. 279, Issue 40, 41839-41845, October 1, 2004
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Microglia-derived Pronerve Growth Factor Promotes Photoreceptor Cell Death via p75 Neurotrophin Receptor*

Bhooma Srinivasan{ddagger}§, Criselda H. Roque{ddagger}, Barbara L. Hempstead¶, Muayyad R. Al-Ubaidi||, and Rouel S. Roque{ddagger}**

From the {ddagger}Department of Cell Biology and Genetics, University of North Texas Health Science Center, Fort Worth, Texas 76107, the Department of Medicine, Weill Medical College of Cornell University, New York, New York 10021, and the ||Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104

Received for publication, March 15, 2004 , and in revised form, July 19, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Reports implicating microglia-derived nerve growth factor (NGF) during programmed cell death in the developing chick retina led us to investigate its possible role in degenerative retinal disease. Freshly isolated activated retinal microglia expressed high molecular weight forms of neurotrophins including that of nerve growth factor (NGF), brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4. Conditioned media from cultured retinal microglia (MGCM) consistently yielded a ~32-kDa NGF-reactive band when supplemented with bovine serum albumin (BSA) or protease inhibitors (PI); and promoted cell death that was suppressed by NGF immunodepletion in a mouse photoreceptor cell line (661w). The ~32 kDa protein was partially purified (MGCM/p32) and was highly immunoreactive with a polyclonal anti-pro-NGF antibody. Both MGCM/p32 and recombinant pro-NGF protein promoted cell death in 661w cultures. Increased levels of pro-NGF mRNA and protein were observed in the RCS rat model of retinal dystrophy. MGCM-mediated cell death was reversed by p75NTR antiserum in p75NTR+/trkA– 661w cells. Our study shows that a ~32 kDa pro-NGF protein released by activated retinal microglia promoted degeneration of cultured photoreceptor cells. Moreover, our study suggests that defective post-translational processing of NGF might be involved in photoreceptor cell loss in retinal dystrophy.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In animal models of degenerative retinal diseases, such as the Royal College of Surgeons (RCS)1 dystrophic rats (1, 2) and light damage retinas (3, 4), photoreceptor cell injury is often accompanied by migration of activated microglial cells into the photoreceptor cell layer and in the subretinal space. The close spatial relationship between the displaced microglial cells and the degenerating photoreceptor cells suggests that microglial cells might be involved in photoreceptor cell death. Activated microglia secrete cytotoxic factors such as free oxygen intermediates, proteases, and excitatory amino acids that may induce neuronal degeneration (5–7). Microglial cells also secrete tumor necrosis factor-{alpha} (TNF-{alpha}) (8), interleukin-1 (9), and tumor inhibitory molecules such as gliastatin 1 (10).

Nerve growth factor (NGF), a member of the neurotrophin family of growth factors that mediate neuronal life and death signals (11), has been implicated in microglia-induced programmed cell death in the developing chick retina (12, 13). Nerve growth factor, synthesized by microglial cells (14–16), has been suggested to promote divergent biological responses because of its interaction with two different receptors, the tropomyosin-related kinase A (TrkA) receptor tyrosine kinase and the p75 neurotrophin receptor (p75NTR). In the absence of TrkA, p75NTR could mediate death signals by formation of ceramide (17, 18) and promotion of Jun kinase activity (19–21).

Support for the cytotoxicity of NGF, however, mostly came from indirect studies involving the use of inhibitors to protect from neurotrophin-induced cell death. Other studies demonstrated that NGF toxicity required preabsorption of NGF to glass beads (3, 13) or supplementation with insulin (22), suggesting that conformational changes to NGF or its receptors might be necessary for the induction of cell death. The toxicity of NGF might also be attributed to the presence of high molecular weight NGF proteins, pronerve growth factor (pro-NGF), in samples of commercial NGF preparations isolated from animal tissues.2 Recent findings of pro-apoptotic activity of neurotrophin precursors, called proneurotrophins, further support the suggestion that NGF toxicity might be attributed to pro-NGF. Lee et al. (23) proposed that proneurotrophins, such as pronerve growth factor (pro-NGF), could be secreted and cleaved extracellularly by matrix metalloproteinases or plasmin to release mature neurotrophins that activate Trk receptors to promote cell survival or differentiation. But under conditions of decreased levels or activity of enzymes, secreted proneurotrophins could bind p75NTR with high affinity and induce p75NTR-dependent apoptosis.

We have previously reported that retina-derived microglial cells secreted soluble products in their conditioned media that promoted apoptosis of photoreceptor cells in vitro (24). With our findings of microglial activation and of increased expression of p75NTR in photoreceptor cells of RCS dystrophic rat retinas (2, 25), and reports of toxicity of microglia-derived NGF-like proteins in programmed retinal cell death, we hypothesized that microglia-derived pro-NGF might be involved in the mechanisms of photoreceptor cell death in degenerative retinal diseases. The following study was done to investigate the secretion of NGF and pro-NGF by retina-derived microglial cells and to determine their toxicity on an established mouse photoreceptor cell line.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Animals—RCS dystrophic rats and age-matched genetic control RCS-rdy+ (Rdy) rats maintained on a 12-hr light/dark cycle with reduced illumination (6–7 foot candles in cages) were sacrificed.3 Animals were injected intraperitoneally with an overdose of sodium pentobarbital, and the eyeballs were enucleated and used for isolation of primary cultures or collection of protein and RNA.

Cell Cultures—Microglial cells were isolated from retinas of 6–8-week-old RCS dystrophic rats and maintained in culture as described (24, 26). Growth medium was supplemented with 200 units of recombinant human macrophage colony stimulating factor (rhMCSF; Genetics Institute, Boston, MA) to maintain the "activated" phenotype of microglial cultures. The expression of microglial markers in the cultures were verified using Bandeiraea (Griffonia) simplicifolia isolectin B4 and antibodies against phosphotyrosine and vimentin prior to use as described (24, 26). The cellular morphology and phagocytosis of fluorescein-labeled beads were used to assess "microglial activation" (24, 26). Cells were used for collection of lysates or conditioned media (CM) within 2 passages from initial isolation. CM was collected from activated microglial cells (MGCM) incubated in serum-free basal medium (BM) consisting of Dulbecco's modified Eagle's medium, 2 mM L-glutamine, 100 units/ml of penicillin, 100 µg/ml streptomycin, and 15 mM Hepes buffer for 48 h in the presence or absence of 0.01–0.05% bovine serum albumin (BSA) or protease inhibitors (PI). The PI used, including aprotinin 1 mg/ml, AEBSF (4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride) 400 µg/ml, leupeptin hemisulfate 1 µg/ml, and pepstatin A 1 µg/ml, were obtained from ICN Biomedicals, Inc., Costa Mesa, CA. The presence of BSA appeared to be necessary to stabilize the activity of the microglia-secreted factors, but it also hampered the purification of the secreted molecules. On the other hand, supplementation with PI was suitable for the purification of the microglia-derived toxic activity, but was not appropriate for biological assays because of direct toxicity of protease inhibitors on the target cultures.

The 661w photoreceptor cell line was used to test the toxicity of the CM.661w cells were derived from transgenic mice retinas expressing SV40T-antigen (27), and have been verified to express photoreceptor cell markers and to exhibit apoptotic pathways observed in animals with retinal degeneration (24, 28, 29). Rat PC12 cells (American Type Culture Collection), known to express neurotrophin receptors, were used as positive controls.

Partial Purification of Pro-NGF—MGCM/PI was concentrated 20-fold using Centricon 3 and fractionated through a Sephadex 75 column using 20 mM PB. Conditioned media was applied to the column and eluted at room temperature at a flow rate of 0.5 ml/min. A total of 25 0.5-ml fractions was collected, pooled into 5 fractions, concentrated, and used for gel electrophoresis and immunoblots or tested for biological activity using MTS assay. The ~32-kDa band in MGCM/PI (MGCM/p32) was partially purified from the active fractions using a mini whole gel eluter (Bio-Rad) according to product specifications. Briefly, fraction II was concentrated using Centricon 3, separated in 4–12% native gradient polyacrylamide minigel, and electroeluted into 14 fractions. Eluted fractions were separated in a 10% SDS-polyacrylamide gel and processed for immunoblot using anti-NGF or anti-pro-NGF, a rabbit polyclonal antibody against GST fusion protein containing amino acids 23–81 of human pro-NGF (30). Fractions containing partially purified MGCM/p32 were pooled and tested for activity on the cultured 661w cells.

Immunodepletion and Neutralization Assays—To determine whether the microglia-derived toxicity was caused by an NGF-related molecule, 661w cells were incubated in NGF-immunodepleted MGCM/BSA. NGF was extracted using a modified immunoprecipitation procedure. Briefly, MGCM/BSA was precleared by incubating in recombinant protein G-agarose beads (rPrG-agarose; Invitrogen) at 4 °C for 1 h then spun at 10,000 x g for 10 min. The supernatant was incubated overnight at 4 °C with 200 ng of affinity-purified rabbit anti-NGF IgG (sc-548; Santa Cruz Biotechnology, Santa Cruz, CA) preadsorbed to rPrG-agarose per 50-µg samples. The next day, the mixture was spun at 10,000 x g for 5 min. The immunodepleted MGCM/BSA was also processed for immunoblot using affinity-purified rat monoclonal anti-NGF IgG (clone 1G3; Promega Corp., Madison, WI) diluted 1:500.

To neutralize p75NTR, a rabbit polyclonal antiserum made against the extracellular domain of the mouse p75NTR (9651; a gift from Dr. Moses Chao, New York University School of Medicine, New York) and shown to block NGF binding to p75NTR (31) was added to the cells at 1:100 dilution 24 h prior to MGCM/BSA treatment.

Cell Survival Assays—The toxicity of MGCM/BSA and the neutralization studies were tested on 661w cells using the MTS assay (Cell Titer 96; Promega Corp.) as described previously (10). Briefly, 661w cells plated at 10,000 cells/well on 96-well plates were incubated in basal medium for 24 h prior to treatment. Following treatment, cells were incubated in 333 µg/ml of MTS and 25 µM phenazine methosulfate for 1 h, and absorbance readings at 490 nm were converted to cell counts based on standard curves generated from 661w cells 4 h from plating. All experiments were done in triplicates. Statistical analyses were done using one-way ANOVA.

Survival assays were also performed on 661w cells using calcein AM and ethidium homodimer (Molecular Probes, Inc., Eugene, OR). Calcein Am is a non-fluorescent cell-permeable dye that is converted to fluorescent calcein by intracellular esterases present in live cells. Ethidium homodimer is a fluorescent non-cell permeable dye normally excluded by live cells but can enter dead cells and bind to nucleic acids. The 661w cells plated on 24-well plates at a density of 25,000 cells/well were incubated under various conditions for 48 h followed by a mixture of 2 µM calcein-AM and 4 µM ethidium homodimer for 45 min and viewed under Olympus inverted microscope with an epifluorescence attachment (Olympus Optical Co., Ltd, Tokyo, Japan).

Reverse Transcription-PCR and Southern Blot Analysis—Total RNA was extracted from cultured cells or mouse retinas for cDNA synthesis and PCR as described (32). PCR primers were designed to amplify p75NTR (GenBankTM accession no. X05137 [GenBank] : 5'-GGAGCCAACCAGACCGTGTG-3', position 288–307 and 5'-CGCCTTGTTTATTTTGTTTGC', position 949–969) and trkA (GenBankTM accession no. M85214 [GenBank] : 5'-TCTCCTTCCCAGCCAGTGTG-3', position 912–931 and 5'-AGGGTTGTCCATAAAAGCAG-3', position 1197–1216). Amplification of 18 S rRNA was used as an internal control. PCR products were run on agarose gels and processed for Southern blot analyses using 32P-labeled probes: 5'-GTGGGCTCGGGACTCGTGTTC-3' for p75NTR or 5'-CCGCCAGCAGGGTGTAGTTC-3' for trkA. Neurotrophin mRNA expression was also determined using RT-PCR. PCR primers were designed to amplify rat NGF (GenBankTM accession no. M36589 [GenBank] .1: 5'-CAGGCAGAACCGTACACAGA-3', position 338–357 and 5'-GTCCGAAGAGGTGGGTGGAG-3', position 567–586); BDNF (GenBankTM accession no. NM_012513 [GenBank] .1: 5'-ATGACCATCCTTTTCCTTACTATGGT-3', position 73–98 and 5'-TCT TCCCCTTTTAATGGTCAGTGTAC-3', position 794–819); NT3 (GenBankTM accession no. NM_031073 [GenBank] : 5'-GATCCAGGCGGATATCTTGA-3', position 186–205 nd 5'-AATCATCG GCTGGAATTCTG-3', position 311–330); and NT4 (GenBankTM accession no. NM_013184 [GenBank] .3: 5'-CTCCTGAGTGGGACCTCTTG-3', position 310–329 and 5'-CACTCACTGCATCGCAC AC-3', position 489–507). PCR products were cloned and sequenced as described (33).

Relative RT-PCR and Southern Blot Analysis—Total RNA was extracted from six 8-week-old RCS and Rdy rat retinas for cDNA synthesis and used for relative PCR as described (32). PCR primers were designed to amplify rat NGF (GenBankTM accession no. M36589 [GenBank] ): (5'-CTCTGTCCCTGAAGCCCACTG-3', position 379–399 and 5'-GCCTGTTTGTCGTCTGTTGTC-3', position 922–942) or rat p75NTR (GenBankTM accession no. X05137 [GenBank] ): (5'-AGCCAACCAGACCGTGTGTG-3', position 290–309 and 5'-TTGCAGCTGTTCCACCTCTT-3', position 933–952); while 18 S competimers (Ambion Inc., Austin, TX) were used to amplify 18 S as internal control. PCR products for NGF and p75NTR were processed for Southern blot analysis using 32P-labeled NGF oligonucleotide probes for NGF (5'-ACCTCCTTGCCCTTGATGTCC-3') and p75NTR (5'-GTGGGCTCGGGACTCGTGTTC-3'). Experiments were done at least three times.

Immunoblotting—Tissues and cells were lysed in modified radioimmune precipitation assay buffer (phosphate-buffered saline, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM sodium chloride, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM aprotinin, and 1 mM sodium orthovanadate). Lysates and concentrated CM were centrifuged at 15,000 x g for 10 min to remove insoluble proteins. Protein concentrations were determined using the BCA Protein Assay kit (Pierce). Aliquots of 25-µg samples were fractionated in 10–12% SDS-polyacrylamide gels together with molecular MASS standards (14.4–97.4 kDa; Bio-RAD) and processed for immunoblotting. Rabbit polyclonal antibodies against NGF (sc-548), BDNF (sc-546), NT3 (sc-547), NT4 (sc-545), and {beta}-tubulin (sc-9104) were obtained from Santa Cruz Biotechnology and used at 1 µg/ml while rabbit anti-pro-NGF IgG (30) was used at 1:1000. Blots were reacted with appropriate horseradish peroxidaseonjugated secondary antibodies (Santa Cruz Biotechnology) and developed using Supersignal West Pico (Pierce). Murine NGF{beta} was obtained from different commercial sources including Alomone Labs, Jerusalem, Israel (N-130), Invitrogen (13257-019), and Promega Corp. (G514B).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Neurotrophin Expression in Primary Cultures of Microglial Cells—To establish the expression of neurotrophins in activated retinal microglia, microglial cells were isolated from RCS dystrophic rat retinas and grown in the presence of rhMCSF to maintain their activated phenotype. Several attempts to isolate similar cultures from age-matched congenic control rats (Rdy) were mostly unsuccessful and often resulted in few flattened cells that did not proliferate and rapidly senesced. Freshly isolated or first passaged microglial cultures were found to stain intensely for B. (Griffonia) simplicifolia isolectin B4, phosphotyrosine, and vimentin (data not shown) as previously observed (26). Cultures were used for isolation of RNA for RT-PCR or for collection of CM in the presence of protease inhibitors (MGCM/PI) to be used for Western blots. Microglial cells expressed PCR products whose sizes 249, 198, 145, and 747 bp were compatible with expected products for rat NGF, NT4, NT3, and BDNF, respectively (Fig. 1A). Specificity of PCR products was confirmed by DNA sequencing. The expression of NGF, NT4, NT3, and BDNF was further investigated using Western blots and showed the presence of ~32, ~55, ~58, and ~36 kDa immunoreactive bands, respectively, in the MGCM/PI (Fig. 1B). The slow migrating neurotrophin bands were much larger than their reported sizes of ~13–14 kDa, and could represent multimeric forms of neurotrophins, neurotrophins bound to soluble truncated receptors (34), or secreted neurotrophin precursors (proneurotrophins) that have not undergone maturation (23). Interestingly, the ~13–14 kDa neurotrophin monomers were never observed in the retinal microglia CM unlike that in the CM of Müller cells (35), the predominant retinal glia.



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  		FIG. 1.
Microglial expression of neurotrophin mRNA and protein. A, primary cultures of rat retina-derived activated microglial cells were tested for expression of NGF, BDNF, NT3, and NT4 using RT-PCR and DNA sequencing. Activated microglial cells generated PCR products of 249, 198, 145, and 747 base pairs consistent with expected sizes of PCR products for rat NGF, NT4, NT3, and BDNF. B, CM were collected from microglial cultures in the presence of protease inhibitors composed of aprotinin 1 mg/ml, AEBSF 400 µg/ml, leupeptin hemisulfate 1 µg/ml, and pepstatin A 1 µg/ml, and processed for immunoblotting using chemiluminescence. Immunoblots of microglial CM showed solitary high molecular mass bands of ~32-, ~55-, ~58-, and ~36-kDa reactive bands for NGF, NT4, NT3, and BDNF, respectively.

 
Microglial Cells Secreted High Molecular Weight Forms of NGF—To begin to investigate the high molecular weight NGF-reactive band secreted by activated retinal microglia, CM were collected from the microglial cultures in the presence or absence of bovine serum albumin (MGCM/BSA) or protease inhibitors (MGCM/PI). The ~32-kDa NGF band was observed in both MGCM/BSA or MGCM/PI but not in MGCM alone. No NGF-reactive bands were observed in any of the CM at the expected size of the mature NGF at ~13 kDa, shown by the commercially obtained NGF{beta} protein (Fig. 2A). These findings suggested that the amounts of ~32 kDa protein and of mature NGF in the unsupplemented CM were either quite low or that they were easily degraded by proteases in the CM. These findings also suggested that BSA and protease inhibitors were able to protect the ~32 kDa band from proteolytic degradation similar to that reported in the case of proneurotrophins (23). The toxicity of microglia-derived products was tested on 661w photoreceptor cells and showed a ~64% decrease in cell counts in cultures treated with MGCM/BSA compared with defined medium alone (BM) (Fig. 2C). MGCM alone or commercial NGF{beta} from various sources also did not exhibit toxicity (p > 0.1).



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  		FIG. 2.
Microglial cells secreted a ~32 kDa NGF reactive protein. A, MGCM collected in the presence of 0.05% bovine serum albumin (MGCM/BSA) or protease inhibitors (MGCM/PI) for 48 h and processed for immunoblot using affinity-purified rabbit anti-NGF IgG at 1 µg/ml displayed a ~32-kDa band, unlike MGCM alone. Commercial NGF{beta} (25 ng) was used as internal control. The blot was developed using chemiluminescence. Light bands observed at ~66 kDa were probably nonspecific since they were absent in other blots. B, concentrated MGCM/BSA was depleted of NGF using a modified immunoprecipitation procedure utilizing 200 ng of affinity-purified rabbit anti-NGF IgG per 50-µg samples. Samples were processed for immunoblot using a rat monoclonal anti-NGF IgG at 2 µg/ml. 661w cells were treated under various conditions for 48 h and assayed for cell survival using MTS/PMS as described under "Experimental Procedures." Experiments were done in triplicate at least five times and subjected to one-way ANOVA followed by Bonferroni's treatment. Bars represent cell numbers (x1000) ± S.D. for each treatment. C, MGCM/BSA but not BM alone, MGCM alone, or NGF{beta} 50 ng/ml promoted decreased cell survival in treated cells. D, NGF immunodepletion inhibited the toxicity of MGCM/BSA on the 661w cells.

 
To further determine the activity of the microglia-derived NGF-reactive bands MGCM/BSA was immunodepleted using anti-NGF IgG. Immunoblots showed ~32-kDa NGF bands in MGCM/BSA and in the immunoprecipitate (pellet), but not in the supernatant (Fig. 2B) consistent with immunodepletion of NGF from the CM. In the presence of MGCM/BSA, the number of surviving cells was reduced to ~42% of that in basal medium (BM) (Fig. 2D). Following treatment with NGF-depleted supernatant (MGCM/BSA-NGF), cell numbers were similar (p > 0.1) to those in BM, suggesting that removal of ~32 kDa NGF protein abolished the toxicity of MGCM/BSA. The number of surviving cells following treatment with NGF-containing beads also resembled that in MGCM/BSA-treated cultures (data not shown). The presence of cytotoxicity only in microglial CM that contained the ~32 kDa protein, and the inhibition of toxicity following its extraction, suggested that the microglia derived toxicity resided in the ~32 kDa protein. Moreover, the presence of the ~32 kDa protein only in MGCM supplemented with BSA or protease inhibitors was consistent with previous suggestions of the protective nature of BSA on the microglia product (24).

~32 kDa NGF Protein Is Pro-NGF—To isolate the ~32 kDa MGCM protein (MGCM/p32), MGCM/PI was fractionated by gel filtration, pooled into 5 fractions (Fig. 3A), and tested for biological activity or immunoreactivity for NGF. Majority of the proteins eluted in fraction III, as verified in silver stained acrylamide gels (data not shown). Treatment of 661w cells with fraction II resulted in significant (p < 0.001) decrease in cell numbers (42% of BM), as in cultures treated with MGCM/BSA (66% of BM)(Fig. 3B). A ~32 kDa band reactive for NGF also separated in fraction II (Fig. 3C). No other fractions exhibited bands reactive for NGF or toxicity to 661w cells.



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  		FIG. 3.
~32 kDa MGCM/BSA protein reacted with anti-pro-NGF IgG. A, to establish the identity and activity of the ~32-kDa NGF-reactive band, MGCM/PI was fractionated using gel filtration column as described under "Experimental Procedures." Collected fractions were pooled into five fractions. B, pooled MGCM/PI fractions were tested for biological activity on cultured 661w photoreceptor cells using MTS/PMS assay and showed toxicity in fraction II similar to MGCM/BSA. Experiments were done in triplicates at least five times and subjected to one-way ANOVA followed by Dunnett's post-test. C, pooled fractions were also assayed using immunoblotting as described under "Experimental Procedures" and showed NGF immunoreactivity only in Fraction II. D, ~32-kDa NGF band was partially purified by electroelution and assayed for immunoblotting using a polyclonal antibody against pro-NGF at 1:1000. NGF{beta} (25 ng) was used as negative control.

 
MGCM/p32 was partially purified from fraction II using electroelution and tested for immunoreactivity to a polyclonal antipro-NGF antibody (30), using NGF{beta} as negative control. The anti-pro-NGF antibody reacted with the partially purified ~32 kDa band (Fig. 3D) but not with NGF{beta}, suggesting that MGCM/p32 was pro-NGF.

Pro-NGF Induces Photoreceptor Cell Death—To further establish that MGCM/p32 was pro-NGF, MGCM/p32, and recombinant furin-resistant pro-NGF (rProNGF) (23) were added to cultured photoreceptor cells and tested for toxicity using calcein AM and ethidium homodimer (Fig. 4). Cells maintained in BM alone appeared flattened and rounded while cells treated with MGCM/p32 or rProNGF were spindle shaped with numerous phase bright and non-adherent cells under phase contrast. The number of cells labeled with ethidium homodimer appeared greater while fewer cells stained with calcein AM, suggestive of increased cell death, in the cultures treated with either MGCM/p32 or rProNGF compared with cells incubated in BM alone. The photoreceptor cell death caused by microglia-secreted products has been attributed to apoptosis in a previous report (24). The above findings support our hypothesis that activated retinal microglial cells secrete ~32 kDa pro-NGF that promotes apoptosis of photoreceptor cells.



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  		FIG. 4.
Pro-NGF induces cell death in cultured photoreceptor cells. To verify the toxicity of pro-NGF on photoreceptor cells, 661w cells cultured in 24-well plates were incubated in BM alone, MGCM/p32, or recombinant pro-NGF 25 ng/ml for 48 h and treated with 2.0 µM calcein-AM and 4.0 µM ethidium homodimer for 45 min to determine cell viability. The apparent greater number of cells stained with ethidium homodimer and fewer calcein AM-labeled cells in cultures treated with either MGCM/p32 or rProNGF are consistent with increased cell death in the cultures.

 
Increased Pro-NGF in Dystrophic Retinas—The expression of the ~32 kDa pro-NGF in vivo was next established using relative RTPCR with Southern blotting and anti-pro-NGF immunostaining of retinas from RCS rats or their genetic controls. Photoreceptor cell injury and death in the RCS retina occurs as early as 2 weeks and are accompanied by migration of activated microglial cells into the outer retina among the photoreceptor outer segments (1, 2). Relative PCR showed increased NGF and p75NTR mRNA expression in RCS compared with age-matched controls (Rdy) (Fig. 5A). Although relative PCR was reflective of the total NGF in the tissues, immunoblots of the same aged retinas with anti-pro-NGF IgG showed specific increases in the ~32 kDa pro-NGF band in RCS retinas compared with Rdy (Fig. 5B).



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  		FIG. 5.
Increased pro-NGF in dystrophic rat retinas. A, expression of NGF and p75NTR mRNA was compared in 8-week-old RCS dystrophic and congenic control (Rdy) rat retinas using relative RT-PCR and Southern blot analysis. 18 S competimers were used to amplify 18 S to normalize sample loading. Southern blots of NGF and p75NTR PCR products showed increased mRNA expression of NGF and p75NTR, respectively, in RCS retinas compared with Rdy. B, pro-NGF levels were determined in RCS and Rdy retinas using immunoblots with anti-pro-NGF antibody at 1:1000 dilution. Microglial lysates (MG) were used as positive control.

 
Cultured Photoreceptor Cells Expressed p75NTR but Not trkA—The expression of p75NTR was also determined in 661w cells using RT-PCR and Southern blot. The 682-bp p75NTR product, but not the 305 bp trkA band, was amplified in 661w cells (Fig. 6A). PC12 cells and normal mouse retinas expressed both p75NTR and trkA. Specificity of PCR products was verified in Southern blots (Fig. 6A, SB). An additional band ~300 bp observed in 661w cells but not in PC12 cells or normal retina did not hybridize with the p75NTR probe. Although the identity of the ~300-bp product remains to be determined, a truncated form lacking three of four extracellular cysteine-rich domains comprising the NGF-binding site in the full-length p75NTR mRNA has been reported in adult mouse brain and spinal cord (36) and appears to be conserved across species including chick, rat, and human. While complete ablation of the full-length and the truncated p75NTR resulted in a severe phenotype characterized by embryonic lethality and significant vascular defects, the exact function of the novel p75NTR spliced variant, its effects on NGF/pro-NGF signaling, and its expression in retinal tissues remain to be investigated.



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  		FIG. 6.
Microglia-derived pro-NGF induced cell death via p75NTR. A, 661w cells were tested for mRNA expression of NGF receptors p75NTR and trkA using RT-PCR and Southern blot (SB). 661w cells expressed p75NTR at low levels but not trkA. Normal mouse (C57BL/6) retinas or PC12 cells were used as internal controls. 18 S was used to normalize sample loading in RT-PCR. B, 661w photoreceptor cells were treated with MGCM/BSA for 48 h. in the presence or absence of the anti-p75NTR antiserum (9651) at 1:100 dilution and assayed for cell survival using MTS/PMS assay as described under "Experimental Procedures." Experiments were done in triplicates at least five times and subjected to one-way ANOVA followed by Bonferroni's treatment. Bars represent cell numbers (x1000) ± S.D. for each treatment. 661w cells were also incubated in basal medium alone (BM) or the antiserum alone as control.

 
To establish the role of p75NTR in the mechanisms of pro-NGF-induced cell death, 661w cells were treated with MGCM/BSA in the presence or absence of a neutralizing rabbit antiserum against p75NTR, and assayed for cell survival using the MTS assay. The number of MGCM/BSA-treated cells was significantly lower (p < 0.001) compared with those in basal medium alone (Fig. 6B). The addition of p75NTR antiserum to the cells prior to MGCM/BSA-treatment resulted in a significant increase in cell counts (p < 0.001) compared with those in MGCM/BSA-treated cultures, suggesting neutralization of the cytotoxic effects of MGCM/BSA on 661w cells. The number of surviving cells treated with p75NTR antiserum alone was similar to that in control.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Neurotrophins such as NGF, BDNF, NT3, and NT4/5 support the survival and differentiation of developing neurons through neurotrophin binding to trk receptors (37, 38). NGF recognizes TrkA; BDNF and NT4/5 bind TrkB; and NT3 binds TrkA, TrkB and TrkC. In addition, all neurotrophins bind p75NTR, enhancing the activation of the trk receptors (37, 39). While NGF primarily exhibits prosurvival and differentiative effects (38), it has also been shown to induce cell death (40–43), and in most studies, p75NTR was involved while TrkA appeared to be absent. These suggest that NGF binding to p75NTR in the absence of TrkA might promote cell death (41). Studies implicating the low affinity neurotrophin receptor p75NTR in apoptotic cell death of neurons and glia (21, 42–45), and findings of increased levels of p75NTR in animals with retinal injury (3, 25, 46), and the absence of Trk receptors in photoreceptor cells (47–49) led us to investigate the role of NGF and p75NTR in photoreceptor cell death in degenerative retinal diseases.

Our study replicates previous findings that activated microglial cells isolated from dystrophic rat retinas secrete soluble products that promote degeneration of cultured photoreceptor cells (24). Although several molecules have been implicated in microglia-derived cytotoxicity (5–10), our study establishes that a ~32 kDa pro-NGF protein secreted by microglial cells targets photoreceptor cells in culture. To our knowledge, this is the first report implicating microglia-derived pro-NGF in the mechanisms of photoreceptor cell death.

Although pro-NGF might also be released by other cell types in the retina, including Müller cells, astrocytes, or RPE cells, our study confirmed that activated retinal microglial cells synthesized and secreted pro-NGF and possibly other proneurotrophins during retinal injury. The presence of high molecular weight bands in the spent medium of microglial cells supported this suggestion. Based on our previous findings of invasion of activated microglial cells into the outer retina during early photoreceptor injury in the dystrophic retina, the increased levels of pro-NGF mRNA and protein in the early dystrophic retina further support pro-NGF secretion by activated microglia. Preliminary data in our laboratory showing pro-NGF immunostaining in activated microglia among degenerating photoreceptor cells in dystrophic retinas2 further support a role for pro-NGF in photoreceptor cell death.

This study also clarified our previous findings of lack of toxicity of MGCM in the absence of BSA (24). The ~32-kDa pro-NGF band was observed in MGCM only when it was supplemented with BSA. Moreover, the intensity of the band appeared to decrease when lower concentrations of BSA were used (data not shown), similar to the decreased toxicity of MGCM with lower BSA concentrations (24). These suggest that BSA might bind the microglial pro-NGF physically, thereby protecting it from adsorption to tissue culture containers or from proteolytic degradation. Microglia are known to secrete numerous proteolytic enzymes that could degrade pro-NGF, and the addition of protease inhibitors to the conditioning medium probably protected pro-NGF from degradation from microglial proteases. However, while plasmin and matrix metalloproteases have been shown to cleave pro-NGF to the mature NGF (23), NGF bands were not observed in the MGCM suggesting that microglial cells might secrete additional proteases that digest pro-NGF and NGF into much smaller peptides. Further studies are needed to better understand the enzymatic processing of pro-NGF and the microglial proteases involved in this process.

Our study also showed that mature NGF was not toxic to cultured photoreceptor cells. This was consistent with the lack of NGF toxicity following intravitreal injections (50) in rodent retinas. Although NGF has been suggested to induce apoptosis of retinal cells in vitro (13), studies often relied on cofactors to elicit NGF toxicity in vitro. For example, Frade and Barde (13) and Allington et al. (51) relied on glass beads, while Frade (22) supplemented with insulin. Other investigators provided indirect evidence of NGF toxicity employing neutralizing antibodies against NGF or its low affinity receptor p75NTR. One could speculate that the apoptotic activity ascribed to NGF in the previous reports might have resulted from pro-NGF activity. Neutralizing antibodies against NGF also recognize pro-NGF while isolates of NGF might be contaminated with pro-NGF. Co-factors might also induce conformational changes to NGF to simulate pro-NGF activation of p75NTR.

Finally, our study supports the growing consensus that the increased expression of p75NTR in the absence of TrkA may be involved in cell death. p75NTR, a member of the TNFR superfamily that includes Fas, TNFR1, and TNFR2, CD27, CD30, CD40, OX40, lymphotoxin-{beta} receptor, and DR3, DR5 (52), contains an intracellular domain that may function for protein-protein interactions and signal transduction through adaptor proteins (53, 54) such as tumor necrosis factor receptor-associated factors (55, 56), neurotrophin receptor-interacting factor (52), p75NTR-associated cell death executor (57), the zinc-finger motif protein SC-1 (58), and neurotrophin receptor-interacting melanoma antigen gene homologue (59).

In the retina, p75NTR has been localized mainly to the retinal ganglion cells and Müller cells (60, 61) of adult rats. In the developing retinas, however, p75NTR has been localized to the neuroblastic layer whose cells undergo programmed cell death. It is tempting to speculate that programmed cell death in the developing retina is regulated by the expression and activity of p75NTR and TrkA and that the loss of p75NTR and TrkA expression protects the developing neurons and push them toward differentiation. It is also tempting to speculate that re-expression of p75NTR in retinal neurons during diseased conditions could mimic their embryonic phenotype predisposing the cells to p75NTR-mediated cell death as we have reported in photoreceptor cells of dystrophic retinas (25). Increased levels of p75NTR staining have also been observed following injury due to light damage (3) and ischemia and reperfusion (62). Although studies by Harada et al. (3) contend that p75NTR was not expressed by photoreceptor cells during light damage, the use of light capture microdissection to collect degenerating cells would not have ascertained that the cells were indeed light-damaged. Moreover, their collection of RNA from the outer nuclear layer would have missed low level mRNA species in the photoreceptor inner segments where protein synthesis, especially of membrane-bound proteins such as p75NTR, is initiated. Last, but not least, the expression of growth factors and receptors such as p75NTR might vary in photoreceptor cells as in other cells depending on the underlying mechanism of cell injury, i.e. light damage versus phagocytic defect in the RCS rat. Clearly, these studies need to be revisited.

In conclusion, we established that the 661w cell line expressed low levels of p75NTR similar to normal photoreceptors cells in the retina. In addition, the increased levels of p75NTR during photoreceptor injury (25) suggested that 661w cells might serve as a good model to study p75NTR signaling pathways in injured photoreceptor cells. The lack of expression of TrkA in 661w cells also confirmed previous reports of absence of trkA mRNA or protein in photoreceptor cells. In the absence of TrkA and increased expression of p75NTR, secretion of pro-NGF by activated microglial cells during retinal disease might facilitate secondary events leading to degeneration of photoreceptor cells. Moreover, the elevated levels of pro-NGF in dystrophic compared with normal retinas suggested that cleavage of pro-NGF might be defective in dystrophic rat retinas. Defective cleavage of pro-NGF, and perhaps, of other proneurotrophins, either because of decreased levels of enzymes or down-regulation of enzyme activity involved in the normal maturational processing of NGF, or increased expression of inhibitors to these proteases could result in accumulation of pro-NGF in the extracellular space and pro-NGF-induced activation of p75NTR signaling pathway. Defective post-translational processing of proneurotrophins could also result in decreased levels of mature neurotrophins required for photoreceptor cell survival. Findings of increased levels of pro-NGF in the parietal cortex of patients with Alzheimer's disease has been proposed to lead to decreased availability of mature NGF to basal forebrain cholinergic neurons (63). Whether this defective processing of pro-NGF contributes directly to cell death in either system remains to be elucidated.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant EY10766 (to R. S. R.), a University of North Texas Health Science Center at Fort Worth Faculty research grant (to R. S. R.), and National Institutes of Health Grant NS30687 (to B. L. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ This work was taken in part from a dissertation submitted to the University of North Texas Health Science Center at Fort Worth in partial fulfillment of the requirements for the degree Doctor of Philosophy. Back

** To whom correspondence should be addressed: Dept. of Cell Biology and Genetics, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, Texas 76107-2699. Tel.: 817-735-5055; Fax: 817-735-2610; E-mail: rroque{at}hsc.unt.edu.

1 The abbreviations used are: RCS, Royal College of Surgeons; BM, basal medium; BSA, bovine serum albumin; CM, conditioned medium; MGCM, microglial conditioned medium; NGF, nerve growth factor; PI, protease inhibitors; pro-NGF, pronerve growth factor; p75NTR, p75 neurotrophin receptor; GST, glutathione S-transferase; ANOVA, analysis of variance; trk, tropomyosin-related kinase; RT, reverse transcriptase; TNF, tumor necrosis factor; BDNF, brain-derived neurotrophic factor; NT, neurotrophin. Back

2 R. Roque, personal observations. Back

3 This was performed in accordance with The ARVO Statement for the Use of Animals in Ophthalmic and Visual Research and Guide for the Care and Use of Laboratory Animals (NIH publication 85-23, revised 1996. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Moses Chao of New York University School of Medicine, New York for the rabbit antiserum to p75NTR and Ramee Lee of Cornell University, New York for providing the recombinant pro-NGF protein and anti-pro-NGF antibody.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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