Microglia-derived Pronerve Growth Factor Promotes Photoreceptor Cell Death via p75 Neurotrophin Receptor
2004; Elsevier BV; Volume: 279; Issue: 40 Linguagem: Inglês
10.1074/jbc.m402872200
ISSN1083-351X
AutoresBhooma Srinivasan, Criselda H. Roque, Barbara L. Hempstead, Muayyad R. Al-Ubaidi, Rouel S. Roque,
Tópico(s)Neuroinflammation and Neurodegeneration Mechanisms
ResumoReports 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. 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. In animal models of degenerative retinal diseases, such as the Royal College of Surgeons (RCS) 1The 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.1The 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. dystrophic rats (1Thanos S. Brain Res. 1992; 588: 21-28Crossref PubMed Scopus (144) Google Scholar, 2Roque R.S. Imperial C.J. Caldwell R.B. Investig. Ophthalmol. Vis. Sci. 1996; 37: 196-203PubMed Google Scholar) and light damage retinas (3Harada T. Harada C. Nakayama N. Okuyama S. Yoshida K. Kohsaka S. Matsuda H. Wada K. Neuron. 2000; 26: 533-541Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 4Ng T.F. Streilein J.W. Invest. Ophthalmol. Vis. Sci. 2001; 42: 3301-3310PubMed Google Scholar), 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 (5Colton C.A. 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Neurobiol. 2000; 10: 381-391Crossref PubMed Scopus (1647) Google Scholar), has been implicated in microglia-induced programmed cell death in the developing chick retina (12Frade J.M. Rodriguez-Tébar A. Barde Y.-A. Nature. 1996; 383: 166-168Crossref PubMed Scopus (666) Google Scholar, 13Frade J.M. Barde Y.-A. Neuron. 1998; 20: 35-41Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). Nerve growth factor, synthesized by microglial cells (14Mallat M. Houlgatte R. Brachet P. Prochiantz A. Dev. Biol. 1989; 133: 322-325Crossref PubMed Scopus (192) Google Scholar, 15Elkabes S. DiCicco-Bloom E.M. Black I.B. J. Neurosci. 1996; 16: 2508-2521Crossref PubMed Google Scholar, 16Heese K. Fiebich B.L. Bauer J. Otten U. Neurosci. Lett. 1997; 231: 83-86Crossref PubMed Scopus (104) Google Scholar), 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 (17Dobrowsky R. Werner M. Castellino A. Chao M. Hannun Y. Science. 1994; 265: 1596-1599Crossref PubMed Scopus (549) Google Scholar, 18Brann A.B. Scott R. Neuberger Y. Abulafia D. Boldin S. Fainzilber M. Futerman A.H. J. Neurosci. 1999; 19: 8199-8206Crossref PubMed Google Scholar) and promotion of Jun kinase activity (19Friedman W.J. J. Neurosci. 2000; 20: 6340-6346Crossref PubMed Google Scholar, 20Yoon S.O. Casaccia-Bonnefil P. Carter B. Chao M.V. J. Neurosci. 1998; 18: 3273-3281Crossref PubMed Google Scholar, 21Casaccia-Bonnefil P. Carter B.D. Dobrowsky R.T. Chao M.V. Nature. 1996; 383: 716-719Crossref PubMed Scopus (718) Google Scholar). 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 (3Harada T. Harada C. Nakayama N. Okuyama S. Yoshida K. Kohsaka S. Matsuda H. Wada K. Neuron. 2000; 26: 533-541Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 13Frade J.M. Barde Y.-A. Neuron. 1998; 20: 35-41Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar) or supplementation with insulin (22Frade J.M. J. Cell Sci. 2000; 113: 1139-1148PubMed Google Scholar), 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. 2R. Roque, personal observations.2R. Roque, personal observations. 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. (23Lee R. Kermani P. Teng K.K. Hempstead B.L. Science. 2001; 294: 1945-1948Crossref PubMed Scopus (1372) Google Scholar) 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 (24Roque R.S. Rosales A.A. Jingjing L. Agarwal N. Al-Ubaidi M.R. Brain Res. 1999; 836: 110-119Crossref PubMed Scopus (94) Google Scholar). With our findings of microglial activation and of increased expression of p75NTR in photoreceptor cells of RCS dystrophic rat retinas (2Roque R.S. Imperial C.J. Caldwell R.B. Investig. Ophthalmol. Vis. Sci. 1996; 37: 196-203PubMed Google Scholar, 25Sheedlo H.J. Srinivasan B. Brun-Zinkernagel A.M. Roque C.H. Lambert W. Wordinger R.J. Roque R.S. Mol. Brain Res. 2002; 103: 71-79Crossref PubMed Scopus (17) Google Scholar), 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. 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. 3This 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. 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 (24Roque R.S. Rosales A.A. Jingjing L. Agarwal N. Al-Ubaidi M.R. Brain Res. 1999; 836: 110-119Crossref PubMed Scopus (94) Google Scholar, 26Roque R.S. Caldwell R.B. Curr. Eye. Res. 1993; 12: 285-290Crossref PubMed Scopus (53) Google Scholar). 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 (24Roque R.S. Rosales A.A. Jingjing L. Agarwal N. Al-Ubaidi M.R. Brain Res. 1999; 836: 110-119Crossref PubMed Scopus (94) Google Scholar, 26Roque R.S. Caldwell R.B. Curr. Eye. Res. 1993; 12: 285-290Crossref PubMed Scopus (53) Google Scholar). The cellular morphology and phagocytosis of fluorescein-labeled beads were used to assess "microglial activation" (24Roque R.S. Rosales A.A. Jingjing L. Agarwal N. Al-Ubaidi M.R. Brain Res. 1999; 836: 110-119Crossref PubMed Scopus (94) Google Scholar, 26Roque R.S. Caldwell R.B. Curr. Eye. Res. 1993; 12: 285-290Crossref PubMed Scopus (53) Google Scholar). 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 (27Al-Ubaidi M.R. Font R.L. Quiambao A.B. Keener M.J. Loiu G.I. Overbeek P.A. Baehr W. J. Cell Biol. 1992; 119: 1681-1687Crossref PubMed Scopus (183) Google Scholar), and have been verified to express photoreceptor cell markers and to exhibit apoptotic pathways observed in animals with retinal degeneration (24Roque R.S. Rosales A.A. Jingjing L. Agarwal N. Al-Ubaidi M.R. Brain Res. 1999; 836: 110-119Crossref PubMed Scopus (94) Google Scholar, 28Krishnamoorthy R.R. Crawford M.J. Chaturvedi M.M. Jain S.K. Aggarwal B.B. Al-Ubaidi M.R. Agarwal N. J. Biol. Chem. 1999; 274: 3734-3743Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 29Tan E. Ding X. Agarwal N. Naash M.I. Al-Ubaidi M.R. Investig. Ophthalmol. Vis. Sci. 2004; 45: 764-768Crossref PubMed Scopus (249) Google Scholar). 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 (30Beattie M.S. Harrington A.W. Lee R. Kim J.Y. Boyce S.L. Longo F.M. Bresnahan J.C. Hempstead B.L. Yoon S.O. Neuron. 2002; 36: 375-386Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar). 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 × 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 × 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 (31Huber L.J. Chao M.V. Dev. Biol. 1995; 167: 227-238Crossref PubMed Scopus (105) Google Scholar) 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 (10Rosales A.A. Roque R.S. Brain Res. 1997; 748: 195-204Crossref PubMed Scopus (26) Google Scholar). 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 (32Jingjing L. Xue Y. Agarwal N. Roque R.S. Investig. Ophthalmol. Vis. Sci. 1999; 40: 752-759PubMed Google Scholar). PCR primers were designed to amplify p75NTR (GenBank™ accession no. X05137: 5′-GGAGCCAACCAGACCGTGTG-3′, position 288–307 and 5′-CGCCTTGTTTATTTTGTTTGC′, position 949–969) and trkA (GenBank™ accession no. M85214: 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 (GenBank™ accession no. M36589.1: 5′-CAGGCAGAACCGTACACAGA-3′, position 338–357 and 5′-GTCCGAAGAGGTGGGTGGAG-3′, position 567–586); BDNF (GenBank™ accession no. NM_012513.1: 5′-ATGACCATCCTTTTCCTTACTATGGT-3′, position 73–98 and 5′-TCT TCCCCTTTTAATGGTCAGTGTAC-3′, position 794–819); NT3 (GenBank™ accession no. NM_031073: 5′-GATCCAGGCGGATATCTTGA-3′, position 186–205 nd 5′-AATCATCG GCTGGAATTCTG-3′, position 311–330); and NT4 (GenBank™ accession no. NM_013184.3: 5′-CTCCTGAGTGGGACCTCTTG-3′, position 310–329 and 5′-CACTCACTGCATCGCAC AC-3′, position 489–507). PCR products were cloned and sequenced as described (33Jingjing L. Srinivasan B. Roque R.S. Angiogenesis. 2001; 4: 103-112Crossref PubMed Scopus (10) Google Scholar). 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 (32Jingjing L. Xue Y. Agarwal N. Roque R.S. Investig. Ophthalmol. Vis. Sci. 1999; 40: 752-759PubMed Google Scholar). PCR primers were designed to amplify rat NGF (GenBank™ accession no. M36589): (5′-CTCTGTCCCTGAAGCCCACTG-3′, position 379–399 and 5′-GCCTGTTTGTCGTCTGTTGTC-3′, position 922–942) or rat p75NTR (GenBank™ accession no. X05137): (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 × 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 β-tubulin (sc-9104) were obtained from Santa Cruz Biotechnology and used at 1 μg/ml while rabbit anti-pro-NGF IgG (30Beattie M.S. Harrington A.W. Lee R. Kim J.Y. Boyce S.L. Longo F.M. Bresnahan J.C. Hempstead B.L. Yoon S.O. Neuron. 2002; 36: 375-386Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar) 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 NGFβ was obtained from different commercial sources including Alomone Labs, Jerusalem, Israel (N-130), Invitrogen (13257-019), and Promega Corp. (G514B). 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 (26Roque R.S. Caldwell R.B. Curr. Eye. Res. 1993; 12: 285-290Crossref PubMed Scopus (53) Google Scholar). 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 (34Zupan A.A. Osborne P.A. Smith C.E. Siegel N.R. Leimgruber R.M. Johnson E.M. J. Biol. Chem. 1989; 264: 11714-11720Abstract Full Text PDF PubMed Google Scholar), or secreted neurotrophin precursors (proneurotrophins) that have not undergone maturation (23Lee R. Kermani P. Teng K.K. Hempstead B.L. Science. 2001; 294: 1945-1948Crossref PubMed Scopus (1372) Google Scholar). Interestingly, the ∼13–14 kDa neurotrophin monomers were never observed in the retinal microglia CM unlike that in the CM of Müller cells (35Taylor S. Srinivasan B. Wordinger R.J. Roque R.S. Mol. Brain Res. 2003; 111: 189-197Crossref PubMed Scopus (89) Google Scholar), the predominant retinal glia. 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 NGFβ 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 (23Lee R. Kermani P. Teng K.K. Hempstead B.L. Science. 2001; 294: 1945-1948Crossref PubMed Scopus (1372) Google Scholar). 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 NGFβ from various sources also did not exhibit toxicity (p > 0.1). 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 (24Roque R.S. Rosales A.A. Jingjing L. Agarwal N. Al-Ubaidi M.R. Brain Res. 1999; 836: 110-119Crossref PubMed Scopus (94) Google Scholar). ∼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. MGCM/p32 was partially purified from fraction II using electroelution and tested for immunoreactivity to a polyclonal antipro-NGF antibody (30Beattie M.S. Harrington A.W. Lee R. Kim J.Y. Boyce S.L. Longo F.M. Bresnahan J.C. Hempstead B.L. Yoon S.O. Neuron. 2002; 36: 375-386Abstract Full Text Full Text PDF PubMed Scopus (375) Google Scholar), using NGFβ as negative control. The anti-pro-NGF antibody reacted with the partially purified ∼32 kDa band (Fig. 3D) but not with NGFβ, 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) (23Lee R. Kermani P. Teng K.K. Hempstead B.L. Science. 2001; 294: 1945-1948Crossref PubMed Scopus (1372) Google Scholar) 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 (24Roque R.S. Rosales A.A. Jingjing L. Agarwal N. Al-Ubaidi M.R. Brain Res. 1999; 836: 110-119Crossref PubMed Scopus (94) Google Scholar). The a
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