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While mature neurotrophins are well-described trophic factors that elicit retrograde survival signaling, the precursor forms of neurotrophins (i.e., proneurotrophins) can function as high affinity apoptotic ligands for selected neural populations. An outstanding question is whether target-derived proneurotrophins might affect neuronal survival/death decisions through a retrograde transport mechanism. Since neurotrophin-3 (NT-3) is highly expressed in non-neural tissues that receive peripheral innervation, we investigated the localized actions of its precursor (proNT-3) on sympathetic neurons in the present study. Pharmacological inhibition of intracellular furin proteinase activity in 293T cells resulted in proNT-3 release instead of mature NT-3 while membrane depolarization in cerebellar granule neurons stimulated endogenous proNT-3 secretion, suggesting that proNT-3 is an inducible bona fide ligand in the nervous system. Our data also indicate that recombinant proNT-3 induced sympathetic neuron death that is p75NTR- and sortilin-dependent, with hallmark features of apoptosis including JNK activation and nuclear fragmentation. Using compartmentalized culture systems that segregate neuronal cell bodies from axons, proNT-3, acting within the distal axon compartment, elicited sympathetic neuron death and overrode the survival promoting actions of NGF. Together these results raise the intriguing possibility that dysregulation of proneurotrophin processing/release by innervated targets can be deleterious to the neurons projecting to these sites.
Proneurotrophins are the precursors of a small family of peptide growth factors that include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4). Until recently, fully processed mature neurotrophins are believed to be the sole ligands responsible for their diverse actions in and outside of the nervous system (Lewin and Barde, 1996; Segal, 2003; Teng and Hempstead, 2003; Gentry et al., 2004). However, both NGF and BDNF precursors can be released as soluble ligands with distinct biological activities (Lee et al., 2001; Nykjaer et al., 2004; Pang et al., 2004; Teng et al., 2005; Woo et al., 2005; Nagappan et al., 2009; Yang et al., 2009a). This supports a paradigm in which proneurotrophins and their mature counterparts interact with distinct co-receptor complexes, thereby inducing diametrically opposite cellular responses. Thus mature neurotrophins activate the Trk receptor tyrosine kinases to elicit well defined signal transduction cascades, while an unrelated p75NTR receptor serves to restrict the fidelity of ligand binding to cognate Trk receptor (Segal, 2003; Teng and Hempstead, 2003; Gentry et al., 2004). In contrast, proNGF and proBDNF engage p75NTR and the vps10p domain-containing receptor sortilin (Nykjaer et al., 2004; Teng et al., 2005; Willnow et al., 2008), with both ligands exerting pro-apoptotic actions on peripheral and central neurons (Lee et al., 2001; Nykjaer et al., 2004; Teng et al., 2005; Volosin et al., 2006; Jansen et al., 2007).
NT-3 was identified as the third neurotrophin family member, based upon sequence conservation with NGF and BDNF (Maisonpierre et al., 1990a). Early in vitro work demonstrated that NT-3 promotes the survival of neuronal sub-populations (Maisonpierre et al., 1990a; Lamballe et al., 1991) and that it is retrogradely transported by both peripheral and central neurons (Distefano et al., 1992), consistent with its role as a target-derived neurotrophic factor. Compared to other neurotrophins, NT-3 exhibits the most widespread distribution in non-neuronal tissues, including many targets of sympathetic and sensory innervations (Schecterson and Bothwell, 1992; Katoh-Semba et al., 1996; Katoh-Semba et al., 1998). In vivo studies of gene targeted animals deficient in NT-3 or it receptor Trk C also support important functions for this ligand in peripheral and central nervous system development (Minichiello and Klein, 1996; Bates et al., 1999; Kahn et al., 1999; Ma et al., 2002; von Bohlen und Halbach et al., 2003). While it is well known that p75NTR modulates the specificity of NT-3 binding to Trk A, B and C (Bibel et al., 1999; Mischel et al., 2001; Kuruvilla et al., 2004), activation of p75NTR alone by NT-3 has also been shown to induce cell death (Friedman, 2000; Wang et al., 2000). Together these findings are consistent with the hypothesis that NT-3 selectively utilizes different receptor complexes to achieve distinct biological endpoints.
Similar to other neurotrophins, NT-3 is synthesized as a high molecular weight precursor (proNT-3) that undergoes furin/proconvertase-mediated cleavage for its release as a mature dimer (Seidah et al., 1996). Interestingly, perturbation of processing results in proNT-3 secretion instead of mature NT-3 (Seidah et al., 1996; Farhadi et al., 2000). Given the study by Ginty and colleagues that NT-3 acts as an intermediate target-derived neuritogenic factor for innervating sympathetic fibers (Kuruvilla et al., 2004), we explored the possibility that locally released proNT-3 might elicit alternative action on sympathetic neuron development and provide evidence for target-derived proNT-3 as a retrograde apoptotic ligand.
HEK 293T cells were maintained in DMEM supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin and 1% pyruvate. Parental PC12 cells and PC12nnr5 (Green et al., 1986) were maintained in DMEM, 10% calf serum, 5% horse serum, 1% penicillin/streptomycin and 1% pyruvate.
Human full length preproNT-3 cDNA was amplified by PCR using primers to introduce a 5′ SacI site with an optimized Kozak consensus for translational initiation, and a heptahistidine (His7) tag, stop codon and BamHI site at the 3′ terminus. In parallel, point mutation of KR to AA (aa137, 138; according to Gene Bank Accession Number: NP_002518) was performed using PCR-based mutagenesis to generate cleavage-resistant proNT-3 cDNA. Constructs encoding native or cleavage-resistant His7-tagged proNT-3 cDNAs, subcloned in pBluescript II SK (pBS NT-3-His7 or pBS proNT-3-His7, respectively), were bidirectionally sequenced. Next recombinant bacculoviral expression vectors encoding native or cleavage-resistant His7-tagged proNT-3 cDNA were generated using the Bac-to-Bac® Baculovirus Expression System by subclonging a PstI-EcoRI insert from pBS NT-3-His7 or pBS proNT-3-His7 into pFastBac I vector. Baculoviral stocks were amplified and propagated using Spodoptera frugiperda (Sf9) cells cultured in Sf-900 II SFM for 72 hours, whereas High Five™ cells cultured in Express Five® SFM were used for protein purification. All baculovirus expression system related reagents and cells were purchased from Invitrogen.
For mammalian expression studies, native and cleavage-resistant preproNT-3 cDNAs were PCR-subcloned into pCMV-Tag 4a vector (Stratagene) using the above described 5′ primer and a separate 3′ primer that removes the NT-3 stop codon to generate C-terminal FLAG-tagged version of both molecules. The resulting constructs were bidirectionally sequenced.
Cellular lysates from baculovirus infected High Five™ cells (5 MOI; 60 hrs post-infection) were prepared in a detergent-free lysis buffer consisting of 50 mM Na-phosphate [pH 7.8], 300 mM NaCl, 20 mM imidazole plus proteinase inhibitors and were used as sources. His7-tagged proNT-3 or mature NT-3 was purified by Ni+ ion chromatography (ProBond® resins) according to manufacturer’s instruction (Invitrogen). Briefly, following washing in 50 mM Na-phosphate [pH 7.8], 300 mM NaCl, 20 mM imidazole, proNT-3-His7 or mature NT-3-His7 was eluted with 50 mM Na-phosphate [pH 7.8], 300 mM NaCl, 500 mM imidazole and were collected in 1-ml fractions. Purification was monitored by Western blot analysis using an anti-NT-3 antiserum that recognizes both proNT-3 and mature NT-3 (SC-547; Santa Cruz Biotechnology) and by silver staining. Fractions containing purified recombinant proteins were pooled and dialyzed against 3 changes of Hank’s Balanced Salt Solution (Invitrogen) and stored in aliquots at −80°C until use. Final concentrations of purified mature NT-3 and proNT-3 were determined by quantitative Western blot using an anti-NT3 antibody (SC-547) against serial dilutions of recombinant NT-3 (Promega) as standards. All experiments were conducted with His7-tagged proNT-3 and His7-tagged mature NT-3 that were purified and processed at the same time to ensure internal consistence.
To compare Trk C activation by NT-3 versus proNT-3, PC12nnr5 cells (Green et al., 1986) were stably transfected with an expression plasmid encoding full length Trk C receptor followed by clonal selection in 500 μg/ml G418. Anti-Trk immunoprecipitation of Trk C-expressing PC12nnr5 clones were carried out with an anti-Trk antiserum (SC-11; Santa Cruz Biotechnology) followed by anti-phosphotyrosine (SC-7020; Santa Cruz Biotechnology) Western blotting. Total cellular lysates were also Western blotted with an anti-pErk antiserum (Ab 9101; Cell Signaling Technology) to corroborate activation of downstream Trk C-dependent signaling or with combined anti-Erk1/2 antisera (SC-93 and SC-153; Santa Cruz Biotechnology) to verify equality of sample loading.
For neuritogenic assay, PC12 cells were transiently transfected with GFP with or without full length Trk C cDNA by Lipofectamine™ 2000 (Invitrogen). Twenty-four hours later, replica cultures were treated with NGF, mature NT-3, or proNT-3 in DMEM containing 0.1% FBS. Paraformaldehyde-fixed, GFP-positive PC12 cells with neurites greater than 2 cell body-diameters in length were scored as positive 48 hrs later. At least 100 cells were counted from randomly chosen fields per culture conditions by an observer blinded to the treatments.
Dissociated SCG neurons were isolated from P0-P2 rats. Alternatively, cultured SCG neurons were prepared from p75NTR null mice (Lee et al., 1992) and wild type control animals. Unless otherwise stated, neurons were plated on laminin-coated Permanox slides and maintained for 7 days in NGF as described (Nykjaer et al., 2004; Teng et al., 2005). On the day of the experiment, replicate cultures were rinsed 5 times with NGF-free medium (MEM, 10% FBS, 0.45% glucose, 2 mM glutamine, 1% pyruvate, 1% penicillin/streptomycin) and treated with either NGF, mature NT-3, proNT-3 or equivalent quantities of diluent (50 mM Na-phosphate [pH 7.8], 300 mM NaCl, 500 mM imidazole dialyzed with 3 changes of HBSS). Where applicable, parallel cultures were concomitantly treated with various antagonists or inhibitors as indicated at the time of ligand addition. After 36-48 hours, SCG cultures were processed for TUNEL analysis (Roche Molecular Biochemicals) and counterstained with anti-neuronal specific beta-tubulin (TuJ1, Covance) and 4′,6′-diamidino-2-phenylindole (DAPI) to visualize nuclei. Apoptotic neurons, identified by TUNEL positivity and/or fragmented nuclei, were scored blinded as to treatment conditions by the observer and at least 200 cells were counted for each culture condition. Where applicable, statistical analyses (Student’s t-test) were performed on the indicated paired samples with significance (p ≤ 0.05) indicates by an asterisk (*). For some experiments, SCG cultures were fixed and stained with phospho-JNK or phospho-c-Jun antibodies (both from Cell Signaling Technology) along with TuJ1 and DAPI. Indirect immunofluorescence analysis was performed using the appropriate secondary antibodies to identify apoptotic neurons.
Dissociated SCG neurons were plated on collagen-coated 35 mm tissue culture plates previously seated with a Teflon divider Camp10 (Tyler Research Instruments) and cultured as described (MacInnis and Campenot, 2002; Ye et al., 2003) with the following modifications. Freshly plated SCG neuron cultures were treated with 5-Fluorodeoxy-Uridine for the first 7 days to eliminate contaminating non-neuronal cells in the center (cell body) compartment. NGF was used at 20 ng/ml (in MEM containing 0.3% methylcellulose, 2.5% FBS, and 0.4% glucose) in the center compartment and at 100 ng/ml (in MEM containing 0.3% methylcellulose, and 0.4% glucose) in the distal axon compartment to induce axonal outgrowth. Seven to ten days later and upon visual inspection of the cultures to ensure axonal growth into the distal compartment, NGF was removed from the center compartment and cultures were treated with 100 ng/ml NGF exclusively in the distal compartment to enrich for neurons that bear processes in the distal compartment. Another 3 days later, NGF within the distal compartment was reduced to 10 ng/ml. Twenty-four hours later, 40 nm orange fluorescent microspheres (Molecular Probe) were added (at 1:1000 dilution in MEM plus 10 ng/ml NGF) to the distal axon chamber. Neurons that were viable with active retrograde transport were thus identified by the uptake of fluorescent microspheres into their cell bodies (Ye et al., 2003). Only replica cultures that were retrogradely labeled were used for further analysis. Where applicable, statistical analyses (Student’s t-test) were performed on the indicated paired samples with significance (p ≤ 0.05) indicates by an asterisk (*).
A GST fusion protein containing the pro-domain of human proNT-3 (aa 21-136) was generated by PCR subcloning the corresponding DNA fragment into the EcoRI-XhoI sites of pGEX6p-1 vector, followed by IPTG induction in bacteria transformant and purification via glutathione-sepharose chromatography. Rabbit antisera (Ab 19573 and 19574) were produced by repeated immunization with the GST-NT-3 pro-domain fusion protein (Pocono Rabbit Farm & Laboratory). High titer antisera (as well as pre-immune control sera from the respective animals) were sequentially purified by negative adsorption to immobilized GST and IgG-enriched by protein A-sepharose chromatography. Further affinity purification of the 19574 antiserum was carried out using conjugated GST-NT-3 pro-domain fusion protein. The resulting antisera (referred to as anti-ProNT-3) were dialyzed in PBS and stored in aliquots at −80°C before further characterization.
Full length human sortilin cDNA was used as a template to generate His6-tagged truncated sortilin lacking the transmembrane and intracellular domain (aa 34-757) and subcloned into the pFastBac I baculoviral vector. Baculoviral stocks were amplified and propagated using Spodoptera frugiperda (Sf9) cells, whereas High Five™ cells cultured in Express Five® SFM were used for protein purification by Ni-ion chromatography. Rabbit anti-sortilin antiserum was obtained by repeated immunization with purified His6-tagged truncated sortilin (Pocono Rabbit Farm & Laboratory) and was further IgG enriched by protein A-sepharose chromatography. Specificity of the anti-sortilin antiserum was verified by Western blotting against lysates of 293 cells transfected with human sortilin cDNA and compared with a commercial monoclonal antibody against sortilin (anti-NTR3; BD Biosciences) (Suppl. Fig. 1).
Initially we surveyed several different published protocols for neurotrophin extraction from tissues (Katoh-Semba et al., 1996; Zhang et al., 2001; Yang et al., 2009b) but have found great variability in how well proNT-3 and mature NT-3 can be extracted. The following procedure was eventually adopted because it affords the most efficient and reproducible means for proNT-3/NT-3 recovery from both neural and non-neural sources: Freshly dissected tissues from rats were immediately processed in ice-cold tissue lysis buffer (20 mM Tris [pH 7.4], 150 mM NaCl, 1% NP-40, 1% Triton X-100, 0.05% SDS, 10% glycerol, 1 mM PMSF and proteinase inhibitor cocktail (Sigma); (Yang et al., 2009b)) using a hand held tissue homogenizer (Omni International), except that hippocampal lysate was obtained by pooling 6-8 dissected hippocampi (kept on dry ice until the end of the dissection) before the homogenization step. Tissue lysates were incubated at 4°C with constant shaking for 15 min and were then cleared by centrifugation at 13,000 x g for 15 min at 4°C before Bradford-based quantitation (Bio-Rad Laboratories). Aliquoted samples (in 1x SDS-PAGE buffer) were boiled and immediately stored at −80°C before further analysis.
Western blotting analysis of proNT-3 and mature NT-3 expression in various tissues was carried out as described (Randolph et al., 2007) using PVDF membrane in 8% non-fat dry milk with a rabbit anti-NT-3 antibody (SC-547; Santa Cruz Biotechnology; 1:1000 dilution). To demonstrate specificity, the anti-NT-3 antiserum was pretreated with 100 fold molar excess of recombinant NT-3 (Promega) for 1.5 hr at 25°C in PBS before using it for Western blotting (Randolph et al., 2007). As a control, anti-NT-3 antiserum that did not receive recombinant NT-3 was processed in parallel before use.
Dissociated P6 cerebellar granule neurons were cultured on poly-L-Lysine-coated 150 mm tissue culture plates and maintained in MEM supplemented with 10% fetal bovine serum, 25 mM KCl, 2 mM GlutaMax™, 0.45% glucose, 1% penicillin/streptomycin and 1% pyruvate. Cultures were also treated with 5-Fluorodeoxy-Uridine for the first 3 days to eliminate contaminating non-neuronal cells. On DIV6, cultured granule neurons were rinsed once with Neurobasal medium (containing 1X B27 supplement, 0.1 mg/ml bovine serum albumin and 0.5 μM GlutaMax™) for 1 hour before treatment with 25 mM KCl to stimulate proNT-3 release. Where indicated, the cell permeable furin inhibitor I (Dec-RVKR-CMK; Calbiochem) was used at 30 μM. To capture secreted proNT-3, a goat anti-NT-3 antiserum (SC-13380; Santa Cruz Biotechnology) was added to the culture medium (1:100 dilution) at the time of treatment. In addition, to prevent proteolytic degradation of proNT-3, a cell impermeant α2 anti-plasmin inhibitor (Calbiochem) was added to all the cultures (Teng et al., 2005). Twenty four hours later, neuronal conditioned media were collected, cleared of cellular debris by centrifugation, and supplemented with the proteinase inhibitors PMSF, leupeptin and aprotinin. Protein A/G agarose beads (Pierce) were then added to the media for 16 hrs to immunoprecipitate proNT-3, followed by three washes in Tris lysis buffer (TLB) containing 20 mM Tris [pH 7.4], 150 mM NaCl, 2mM CaCl2 and 10% glycerol. To elute captured proNT-3, washed agarose beads were incubated with 0.1M glycine [pH 2.5] for 10 minutes at 4°C. Eluates were immediately neutralized with 1.5 M Tris [pH 8.8] before addition of 5 X SDS-PAGE sample buffer, followed by Western blotting with anti-NT-3 or anti-proNT-3 antisera.
HEK 293T cells were transfected with mammalian expression vectors encoding p75NTR, and/or Myc-tagged sortilin in the presence or absence of FLAG-tagged cleavage-resistant proNT-3. Forty-eight hours later, cells were harvested in lysis buffer containing 20 mM Tris [pH 7.4], 150 mM NaCl, 1% NP-40 supplemented with proteinase inhibitors. To detected intracellular proNT-3 interaction with sortilin, anti-FLAG immunoprecipitates were Western blotted with a rabbit anti-Myc antibody (Abcam) according to standard protocol.
To detect p75NTR and sortilin binding to exogenously added proNT-3, 293T cells expressing both, or either, receptor were treated for 1 hr at 37°C with conditioned medium (MEM containing 2 mM GlutaMax™, 0.45% glucose, 1% penicillin/streptomycin and 1% pyruvate) from 293T cells transfected with FLAG-tagged proNT-3 cDNA. Receptor expressing cells were then harvested in TLB before overnight immunoprecipitation with a biotinylated goat anti-p75NTR antibody (BAF367; R&D Systems) using strepavidin beads (Pierce). After five washes in TLB, the receptor-bound proNT-3 was eluted with 0.1M glycine [pH 2.5] and processed as described above for Western blotting analysis.
Previous work demonstrated that the precursors of NGF and BDNF (i.e., proNGF and proBDNF, respectively) are secreted ligands with opposing biological actions to their mature neurotrophin counterparts. During development, NT-3 exhibits the highest level of expression among the neurotrophins examined in a variety of target tissues (Maisonpierre et al., 1990b; Katoh-Semba et al., 1996; Katoh-Semba et al., 1998). Yet it remains unclear whether NT-3 is released as a higher molecular weight form (i.e., proNT-3), and if so, how proNT-3 affects neuronal functions. Therefore we first sought evidence of endogenous proNT-3 secretion in the present study.
Rabbit antisera, raised against the pro-domain of human prepro-NT-3 (referred to as anti-ProNT-3), were assessed for reactivity towards proNT-3 but not mature NT-3 or other proneurotrophins. As shown in Fig. 1A, Sf9 cells infected with a full-length preproNT-3 containing viral vector express both mature NT-3 (~14.5 kDa) and the high molecular weight proNT-3 (~32 kDa); based on Western blot analysis using a commercial anti-NT-3 antiserum that recognizes the C-terminal portion of both NT-3 and proNT-3. Reprobing the membrane with our anti-proNT-3 antiserum revealed only the 32 kDa proNT-3 species. The specificity of the anti-proNT-3 antiserum was confirmed by Western blot analysis of lysates from insect cells expressing either proNGF, proBDNF or proNT-3. These proteins were generated from expression constructs in which the furin consensus sites of the neurotrophins were mutated to block cleavage ((Negro et al., 1994; Seidah et al., 1996; Lee et al., 2001; Teng et al., 2005) and Fig. 3A below). As shown in Fig. 1B, immunopositive signal was only detectable from cells that expressed proNT-3. Reprobing the blot with anti-NGF and anti-BDNF antisera confirmed the presence of proNGF and proBDNF, respectively, in the corresponding cell lysates (data not shown). Consistent with the results from Fig. 1B, proNT-3 antiserum specifically recognizes HEK 293 cells that were transfected with proNT-3, but not proNGF or proBDNF, expression plasmid by immunofluorescence microscopy (Fig. 1C). These results together indicate that our antiserum specifically recognizes the pro-domain of proNT-3 with no detectable cross-reactivity to mature NT-3 or other proneurotrophins.
Multiple tissues from newborn rat contain proNT-3 and mature NT-3 at varying abundance and ratios as assessed by anti-NT-3 Western blot analysis (Fig. 2A). The specificity of proNT-3 and mature NT-3 immunoreactivity was confirmed by probing replica blot with the same anti-NT-3 antiserum in the presence of excess recombinant NT-3 (Fig. 2B). Although we cannot rule out the possibility that lower molecular weight proNT-3 isoforms that were present in some of the tissues arose from partial proteolytic degradation during sample preparation, it is noteworthy that proNT-3 of various sizes (~28-37 kDa) have been predicted to exist due to alternative 5′ exon usage as well as multiple in-frame ATG start sites within the NT-3 transcripts (Leingartner and Lindholm, 1994; Kendall et al., 2000).
Interestingly, representative CNS tissues of P1 rat appear to express predominantly the high molecular weight proNT-3. Although prolonged exposure to film did reveal trace amount of mature NT-3 in these CNS tissues (data not shown), it is apparent that the ratio of proNT-3 to mature NT-3 is significantly greater in the developing CNS, compared to visceral organs of the same age. In contrast, mature NT-3 is readily detectable in the adult brain. As shown in Fig. 2C, both proNT-3 and mature NT-3 are present in the cerebellum of older animals, consistent with a recent finding that the relative expression of proBDNF and mature BDNF is developmentally regulated (Yang et al., 2009b).
Past studies have documented high level of NT-3 expression in the cerebellum (Lindholm et al., 1993; Katoh-Semba et al., 1998) and activity-dependent release of NT-3 from cultured cerebellar granule neurons (Sadakata et al., 2004; Sadakata et al., 2007). These ELISA-based assays, however, did not distinguish whether proNT-3 or mature NT-3 is secreted. Since cerebellum expresses mostly proNT-3 at early postnatal age (Fig. 2A), we carried out anti-NT-3 immunoprecipitation analysis from conditioned media of KCl-depolarized cultured cerebellar granule neurons (Fig. 2D) to directly examine whether proNT-3 is released in an activity dependent manner. Our data indicate that under basal culture condition, there was negligible proNT-3 release from P6 granule neurons. However, upon membrane depolarization, a significant amount of proNT-3 can be detected in the media of these cultures.
Previous work utilized the baculoviral-insect cell system to purify biologically active mature NT-3 and has provided indirect evidence of R138:Y139 as the site of proNT-3 cleavage to yield mature NT-3 (Negro et al., 1994). In order to assess the potential roles of proNT-3 within the nervous system, we generated a recombinant proNT-3 mutant in which the putative furin/proconvertases cleavage motif has been mutated (K137R138→A137A138). As shown in Fig. 3A, HEK 293T cells transfected with epitope-tagged wild type preproNT-3 cDNA were able to process and release mature NT-3 into the culture medium. Consistent with past findings (Seidah et al., 1996; Farhadi et al., 2000), inhibition of intracellular furin/proconvertase activity by Dec-RVKR-CMK resulted in the secretion of ~35-37 kDa proNT-3 instead of mature NT-3. Importantly, we found that the K137R138→A137A138 mutation completely prevented the conversion of proNT-3 to the mature species. Therefore K137R138 appear to be the critical residues that are required for proNT-3 processing to mature NT-3 in mammalian cell system. We also demonstrated that furin inhibitor only modestly augmented proNT-3 secretion from cultured cerebellar neurons in the absence of membrane depolarization (Fig. 3A; left panel), consistent with the data in Fig. 2 that proNT-3 is the predominant NT-3 isoform in early postnatal CNS neurons.
Taking advantage of the above findings, baculoviral vectors harboring wild type or furin cleavage mutant preproNT-3 cDNA with an in-frame C-terminal His7 tag were used for mature NT-3 or proNT-3 production, respectively, in the insect High Five™ cells. Following Ni-ion chromatography and anti-NT-3 Western blot analysis (Fig. 3B), purified mature His7-tagged NT-3 was detectable at the predicted molecular weight (~14 kDa) with the minor presence of a higher molecular species (~30 kDa); presumably a small fraction of proNT-3 has escaped cleavage due to overexpression (Negro et al., 1994). By contrast, eluates from cells expressing the furin cleavage proNT-3 mutant contain exclusive the ~30 kDa proNT-3 species (Fig. 3C) that was recognized by the anti-proNT-3 antibody (data not shown).
To assess the biological actions of proNT-3, we examined whether recombinant proNT-3 exhibits similar activity as mature NT-3 in mediating TrkC-specific signal transduction. A PC12 mutant cell line which does not express the NGF-responsive Trk A receptor (PC12nnr5; (Green et al., 1986)) was stably transfected with full length TrkC. As shown in Fig. 4A, treatment of the TrkC-expressing PC12nnr5 cells with NT-3, but not proNT-3, resulted in TrkC tyrosine phosphorylation as well as in p42/p44 MAP kinase activation. As expected, NGF did not elicit downstream signaling in these cells. Consistent with the data from Fig. 4A, transient expression of TrkC in wild type (NGF-responsive) PC12 cells imparts NT-3 responsiveness (Fig. 4B). In contrast, proNT-3 treatment did not induce significant neurite extension in TrkC-expressing PC12 cells. As positive controls, NGF-elicited neurite outgrowth was observed in GFP-transfected or TrkC-transfected PC12 cells. Together these data suggest that proNT-3 does not promote neuronal differentiation via TrkC.
As past studies have demonstrated that high concentrations (50-100 ng/ml; i.e., 1-2 nM) of mature NT-3 triggered apoptosis in a p75NTR dependent manner in hippocampal neurons (Friedman, 2000) and a smooth muscle cell line (Wang et al., 2000), we next asked whether proNT-3 can induce cell death and if so, which receptor/s are required for this action. Sympathetic neurons from the superior cervical ganglia (SCG) have been a well-characterized model system for analysis of p75NTR-mediated apoptotic signaling (Bamji et al., 1998; Palmada et al., 2002; Kenchappa et al., 2006). To examine whether proNT-3 induces SCG neuron death, replica cultures of rat sympathetic neurons were deprived of NGF in the presence or absence of recombinant cleavage resistant proNT-3. Assessment of TUNEL positivity and nuclear morphology revealed that proNT-3, at subnanomolar concentrations, significantly increased the proportion of neuronal apoptosis when compared to non-proNT-3 treated (but NGF-deprived) controls (Fig. 5A). Consistent with the finding that sympathetic neuron survival per se is NT-3 independent (Kuruvilla et al., 2004), mature NT-3, when applied at identical concentration to that of proNT-3 (0.1 nM), did not enhance or reduce apoptosis of the NGF-deprived cultures.
We also found that subnanomolar concentrations of NGF effectively rescued proNT-3 treated SCG neurons from dying, suggesting that activation of the Trk A receptor in these cells can counter the apoptotic actions of p75NTR; consistent with past findings in NGF-treated oligodendrocytes (Yoon et al., 1998) and in proBDNF-treated sympathetic neurons (Teng et al., 2005). These data are in contrast, however, to the study in basal forebrain neurons in which proneurotrophin elicited death was not abrogated by Trk receptor activation (Volosin et al., 2006). The underlying mechanism that differentiates these cell type specific responses is presently unclear.
Having identified proNT-3 as a pro-apoptotic ligand, we further compared the relative effectiveness of proNT-3 with other previous described triggers of sympathetic neuron death. Our data demonstrate that proNT-3, at comparable concentrations (0.1 nM), is as effective as proNGF in inducing SCG apoptosis (Fig. 5C; (Nykjaer et al., 2004)). Likewise, under mildly depolarizing condition, high concentration of BDNF (2 nM; (Bamji et al., 1998)) and proNT-3 (0.1 nM) were able to promote SCG neuron death to similar extent (Fig. 5D).
The data from Fig. 5 are consistent with a role for proNT-3 as a high affinity neuronal apoptotic ligand. Its biological effect is thus reminiscent of the recently identified dual receptor mechanism for proneurotrophin actions (Lee et al., 2001; Nykjaer et al., 2004; Teng et al., 2005; Jansen et al., 2007). We therefore considered whether both p75NTR and sortilin, are required for proNT-3 apoptotic actions. As shown in Figure 6A, co-expression of FLAG-tagged proNT-3 with Myc-tagged sortilin in HEK 293T cells resulted in complex formation between these two components as detected by anti-FLAG immunoprecipitation and anti-Myc Western blot analysis. Surprisingly, we were unable to detect a stable complex between p75NTR and proNT-3 nor did the co-expression of sortilin augment p75NTR-proNT-3 interaction by co-immunoprecipitation analysis from cellular lysates, suggesting that the biosynthetic pathways of ectopically expressed p75NTR and proNT-3 do not overlap. Therefore, to functionally assess whether both p75NTR and sortilin are required for proNT-3 binding, we transfected these two receptors, alone or in combination, in 293T cells followed by exogenous proNT-3 treatment. As shown in Fig. 6B, we were able to detect proNT-3 as well as sortilin in anti-p75NTR immunoprecipitates from cells that co-express both p75NTR and sortilin. In addition, NT-3 immunoreactivity was detectable in lysate of these cells as multiple partially cleaved forms, suggesting that proNT-3 binding to p75NTR (and presumably internalization and subsequent degradation) requires sortilin co-expression.
To directly assess the role of p75NTR in proNT-3-elicited neuronal apoptosis, we compared the effects of proNT-3 on SCG cultures derived from p75NTR null mice versus wild type control animals (Fig. 6C). Consistent with the data obtained with rat SCG, proNT-3 induced an approximately 50% increase in neuronal apoptosis of wild type mouse SCG cultures. In contrast, treatment of p75NTR-null SCG neurons with proNT-3 did not enhance cell death, indicating that proNT-3 induced apoptosis occurred in a p75NTR-dependent manner.
To evaluate whether sortilin is required for proNT-3 induced SCG neuron death, we took advantage of the finding that neurotensin competes with proneurotrophins for sortilin binding (Nykjaer et al., 2004; Teng et al., 2005; Quistgaard et al., 2009) but does not promote survival of NGF-deprived sympathetic neurons (Unsicker and Stogbauer, 1992). Thus we compared the effects of proNT-3 on SCG neuron survival in the absence or presence of 20 μM neurotensin. As shown in Fig. 6D, neurotensin effectively abrogated the pro-apoptotic effect of proNT-3 but did not otherwise display additional survival promoting action, consistent with past findings (Unsicker and Stogbauer, 1992; Nykjaer et al., 2004; Teng et al., 2005). Furthermore, an antibody raised specifically against the ecto-domain of sortilin was able to block proNT-3 induced cell death (Suppl. Fig. 1 and Fig. 6E). Taken together, these data suggest that proNT-3 elicits apoptotic signaling via p75NTR and the co-receptor sortilin.
Past studies on p75NTR-mediated signaling identified a number of downstream pathways that play critical roles in cellular apoptosis (Dechant and Barde, 2002; Roux and Barker, 2002; Kenchappa et al., 2006; Volosin et al., 2008). Among them, the stress-induced MAP kinase member JNK has been well-described as a p75NTR-inducible apoptotic kinase (Harrington et al., 2002; Linggi et al., 2005). Since proNT-3 appears to require p75NTR for its apoptotic actions (Fig. 6B), we questioned whether JNK might be a downstream effector of p75NTR activation by proNT-3. Since NGF deprivation alone results in SCG neuron death and JNK activation (Eilers et al., 1998), the additive effect of proNT-3 on JNK activation, if any, might be masked in such an experimental paradigm. Membrane depolarization prevents sympathetic death upon trophic factor withdrawal (Franklin et al., 1995). We therefore asked whether proNT-3 can still induce neuronal apoptosis when NGF-deprived cultures were maintained by KCl. As shown in Fig. 7A and consistent with past findings, chronic membrane depolarization effectively promotes neuronal survival in the absence of NGF. Importantly, proNT-3, but not mature NT-3, remained effective in triggering SCG apoptosis even under depolarizing condition.
Using this tissue culture paradigm, we assessed whether proNT-3 activates JNK in sympathetic neurons. Replica cultures of NGF-deprived but KCl-maintained neurons were treated with either NGF, NT-3 or proNT-3, or no additive as indicated. Total cellular lysates were harvested 4 hours after treatment and were analyzed by Western blotting analysis using antisera that differentiate survival or apoptotic signaling (Fig. 7B). As expected, NGF, but not NT-3 or proNT-3, stimulated tyrosine phosphorylation of Trk A. In contrast, proNT-3, but not NGF or NT-3, augments JNK phosphorylation, consistent with its apoptotic effects. In concert with the Western blot data, proNT-3 treated neurons were strongly positive for p-JNK and p-Jun by immunofluorescent analysis (Fig. 7C). Significantly, the JNK inhibitor SP600125 completely prevented proNT-3 induced SCG death, suggesting a causal role for JNK as downstream apoptotic effector (Fig. 7D). As expected from past findings (Harris et al., 2002; Xu et al., 2003), inhibition of JNK activity was found to rescue NGF deprived sympathetic neurons from dying.
Taken together, our data from Figs. Figs.55--77 have identified JNK as a signaling component downstream of proNT-3 induced cell death and support the hypothesis that proNT-3 utilizes p75NTR to initiate neuronal apoptotic cascade. Unlike that reported for mature NT-3 (Friedman, 2000; Wang et al., 2000), sub-nanomolar concentrations of proNT-3 are sufficient to activate apoptotic signaling. Therefore proNT-3 appears to be a high affinity pro-apoptotic ligand for the sympathetic neurons.
Retrograde survival signaling is a hallmark feature of the neurotrophin hypothesis (Zweifel et al., 2005; Ibanez, 2007). Although NT-3 is not effectively taken up at the axonal termini of young SCG neurons (Kuruvilla et al., 2004), anti-NT-3 immunoreactivities in peripheral tissues, including targets of sympathetic innervation, far exceed that in the nervous system ((Kaisho et al., 1994; Katoh-Semba et al., 1996); and Fig. 2A). In non-neuronal cell lines, inhibition of proNT-3 cleavage can result in its release instead of mature NT-3 ((Seidah et al., 1996; Farhadi et al., 2000); and Fig. 3). We therefore considered the possibility that proNT-3, if secreted from target tissues, might be deleterious to the ganglia innervating these sites.
Retrograde neuronal signaling can be studied in vitro in compartmentalized cultures where the neuronal cell bodies are segregated from the distal axons (Fig. 8A; (MacInnis and Campenot, 2002; Ye et al., 2003)). To test whether proNT-3 can elicit neuronal cell death from the distal axons, we selectively applied proNT-3 or mature NT-3 to this compartment while neuronal cell bodies continued to receive NGF for trophic support. Conversely, we treated distal axons with NGF but cell bodies with proNT-3 or NT-3. Neuronal death was assessed 2 days later by the presence of condensed or fragmented nuclei as revealed by DAPI staining following published techniques (Fig. 8B; (Ye et al., 2003)). Consistent with past findings, NGF applied to the center and distal compartments maintained neuronal survival (MacInnis and Campenot, 2002; Ye et al., 2003). By contrast, simultaneous treatment of both compartments with proNT-3 elicited cell death, consistent with our mass culture data above. Mature NT-3, when used at low concentration (10 ng/ml), did not elicit any appreciable survival promoting or apoptotic effect. Intriguingly, we found that proNT-3, applied to the distal axons alone, effectively induced sympathetic neuron death even when neuronal cell bodies continued to receive trophic support from NGF (Fig. 8C). These observations therefore suggest that proNT-3 can initiate apoptotic signaling locally which propagates back to the neuronal cell bodies .
It is also important to note that NGF applied to the distal axons did not rescue sympathetic neuron death that was triggered by proNT-3 present exclusively in the cell body compartment (Fig. 8C). Under this paradigm, neuronal cell bodies continued to receive trophic support from the distal NGF-treated axons both before and during proNT-3 application to the cell bodies. Therefore, it is unlikely that the survival signal from distally applied NGF did not arrive “in time” to rescue these neurons. These results suggest that the retrograde survival promoting effects of NGF can be overcome by local apoptotic signal located at the cell body.
The roles of proNT-3 have not been extensively studied. Using recombinant proNT-3 in which the furin/proconvertase cleavage consensus has been mutated to prevent the conversion to mature NT-3, we report here that proNT-3 is a pro-apoptotic ligand for specific neural populations such as SCG (Fig. 5) and DRG (Suppl. Fig. 2). This action requires the co-receptor complex of p75NTR and sortilin and downstream JNK signaling (Fig. 7); similar to how proNGF and proBDNF induce neuronal apoptosis (Nykjaer et al., 2004; Teng et al., 2005; Volosin et al., 2006). Interestingly, γ-secretase dependent cleavage of p75NTR has been shown as a causal trigger for proBDNF-induced sympathetic neuron death (Kenchappa et al., 2006). Future studies are needed to determine if all proneurotrophins or neurotrophins utilize a similar p75NTR-dependent mechanism for signal transduction (Kenchappa et al., 2006; Bertrand et al., 2008; Vilar et al., 2009a; Vilar et al., 2009b). The complexity of neurotrophin signaling through p75NTR, however, can be gleaned from early binding and mutagenesis studies (Rodriguez-Tebar et al., 1992; Ryden et al., 1995; Urfer et al., 1995), as well as recent crystal structures of NGF:p75NTR and NT-3:p75NTR (He and Garcia, 2004; Gong et al., 2008). One conclusion from these collective works is that there are significant differences between two structurally related ligands in their interactions with p75NTR. Presumably the binding of the neurotrophin pro-domain to sortilin will further influence how p75NTR signals during normal development (Woo et al., 2005; Nakamura et al., 2007; Singh et al., 2008) and in pathological conditions (Volosin et al., 2006; Al-Shawi et al., 2008).
A surprising aspect of our study is the finding that proNT-3 can induce neuronal death when it is present exclusively at the distal axons, implicating some form of retrograde death signaling to the soma. Prior analysis of TrkA-dependent trophic signaling suggests that specific cellular events can be triggered by NGF at the distal axons which propagate back to the neuronal cell bodies (Howe and Mobley, 2005; Zweifel et al., 2005). Whether proNT-3 recruits distinct cellular components for its retrograde apoptotic actions, as has been recently demonstrated for sympathetic neurons upon NGF deprivation at the axonal termini (Mok et al., 2009), remains an intriguing possibility. Nevertheless, it is interesting to note that NGF effectively rescued proNT-3-treated neuron from death in mass culture (Fig. 5B) but was unable to prevent SCG apoptosis when presented in distinct compartment from proNT-3 (Fig. 8C). These observations therefore suggest that the ability of NGF to “terminate” proNT-3-initiated apoptotic cascade depends on whether pro-survival signaling (presumably via TrkA activation) occurs in the same membrane proximal location.
Sympathetic neurons of the superior cervical ganglia do not retrogradely transport NT-3 (Distefano et al., 1992; Reynolds and Hendry, 1999; Kuruvilla et al., 2004). Rather NT-3 acts via Trk A locally on nascent axons to promote process outgrowth (Kuruvilla et al., 2004). Thus our finding that proNT-3 elicits apoptotic signaling from the distal axons distinguishes this ligand with the localized effects of mature NT-3 in biological significance. Specifically, since perturbation of proNT-3 processing results in proNT-3 release instead of mature NT-3 (Fig 3A; (Seidah et al., 1996; Farhadi et al., 2000)), it is conceivable that neurodegeneration can be caused by proNT-3 secretion, perhaps in addition to mature neurotrophins deprivation, from innervated targets. Given that multiple targets of peripheral innervation express high levels of NT-3 (Kaisho et al., 1994; Katoh-Semba et al., 1996), this paradigm might have broad implications in the etiology of neurodegenerative diseases that involve neurotrophin processing and release (Lessmann et al., 2003; Srinivasan et al., 2004; Bruno and Cuello, 2006; Domeniconi et al., 2007). Accordingly the finding of proBDNF accumulation at sites of distal nerve crush (Wang et al., 2006) is consistent with the hypothesis that retrograde transport of proneurotrophins is a distinct cellular event upon injury.
While our data suggest that proNT-3 is an apoptotic ligand for peripheral neurons, we also documented activity-dependent endogenous proNT-3 secretion from cerebellar granule neurons. Since it is well known that membrane depolarization promotes cerebellar granule neurons survival (D’Mello et al., 1993), proNT-3 might play a distinct, non-apoptotic, role in the developing CNS. Indeed the findings that proBDNF acts through p75NTR to induce hippocampal LTD (Woo et al., 2005) and synaptic retraction at the neuromuscular synapses (Yang et al., 2009a) are consistent with such a possibility. Alternatively, since both secreted proNGF and proBDNF can be cleaved by extracellular proteinases (Pang et al., 2004; Bruno and Cuello, 2006) as well as by glia-dependent uptake and re-released as mature neurotrophins (Althaus and Kloppner, 2006; Bergami et al., 2008), proNT-3 might be subjected to similar mechanisms for localized processing to mature NT-3 in order to limit TrkC activation. Our finding that TrkC-expressing PC12nnr5 cells remained somewhat responsive to proNT-3 is consistent with a model of proneurotrophin processing upon endocytosis (Boutilier et al., 2008). Although our data indicate that mature NT-3 is present in greater abundance in the adult cerebellum (Fig. 2C), and are therefore consistent with a crucial role for TrkC in CNS development (Minichiello and Klein, 1996; von Bohlen und Halbach et al., 2003), future study will be required to understand the physiological significance of proNT-3 processing and release in relation to p75NTR vs TrkC activation throughout development and in adulthood.
Past analysis has identified the dual roles of sortilin as an intracellular neurotrophin sorting molecule (Chen et al., 2005) and an extracellular proneurotrophin receptor (Nykjaer et al., 2004; Teng et al., 2005). Our findings are consistent with the latter work and implicate sortilin as a p75NTR co-receptor for proNT-3. Given the observation that proNT-3 pro-domain cleavage can be uncoupled from the secretory pathway (Figs. (Figs.22 and and3;3; (Seidah et al., 1996; Farhadi et al., 2000)), elucidating the molecular interactions between proNT-3 and sortilin will be crucial to understand whether sortilin also modulates proNT-3 processing and release. Although sortilin is but one of several related Vps-10p domain-containing molecules (Hermans-Borgmeyer et al., 1998; Mazella, 2001; Hermey et al., 2004), our data using a sortilin antagonist (Fig. 6D) and an anti-sortilin blocking antiserum (Fig. 6E) strongly suggest that the apoptotic action of proNT-3 requires sortilin. Consistent with these findings, proNT-3 uptake in a surrogate cell line can only be re-constituted in the presence of sortilin and p75NTR (Fig. 6B). However we cannot rule out the possibility that in vivo where both p75NTR and multiple sortilin family members are expressed at varying abundance and in different cell types, the specificity of proneurotrophin actions might be mediated by additional sortilin family members.
In summary, our study raises the intriguing possibility of proNT-3 as a target-derived apoptotic factor. Along with the reported role of truncated Trk C in mediating mature NT-3 actions (Esteban et al., 2006), NT-3 and its precursor proNT-3 are capable of eliciting a plethora of biological events that differ in terms of co-receptor utilization and mechanisms of downstream signaling, consistent with their widespread expressions in both neural and non-neural tissues.
Supplementary Figure 1. Specificity of anti-sortilin antiserum Ab 727. Purified His6-tagged soluble sortilin and lysates from 293T cells transiently transfected with either GFP (as negative control) or full length human sortilin cDNA were Western blotted with preimmune serum, a commercial anti-sortilin monoclonal antibody (α-NTR3; BD Biosciences) or Ab 727 (Materials and Methods) as indicated. Numbers on the left indicate positions of the molecular weight markers.
Supplementary Figure 2. ProNT-3 induces DRG neuron apoptosis. E16 rat dorsal root ganglionic neurons were cultured in 100 ng/ml NGF. Seven days later, replica cultures were washed free of NGF (None) and were treated with 10 ng/ml NGF, equal molar of NT-3 or proNT-3 (i.e., 2 ng/ml NT-3 or 4 ng/ml proNT-3) as indicated. Percentages of apoptotic DRG neurons were determined as described for SCG neurons (Fig. 4B). The data represent results from three independently conducted experiments and vertical errors represent SEM.
We are grateful to Dr. Barbara Hempstead for her advice and generous support. We thank Dr. Francis Lee for helpful discussion, Dr. Robert Campenot for sharing his expertise on compartmentalized culture system, and Drs. Anders Nykjaer and Claus Munck Petersen for communicating their unpublished data. We also thank Taeho Kim for his advice on proneurotrophin immunoprecipitation. This work is supported in part by the NIH (NS21072 and HD23315 to M.V.C.; and NS057627 to K.K.T.) and the James Birrell Neuroblastoma Fund at the Hospital for Sick Children (to R.T.; R.T. was at Weill Cornell Medical College at the time of the study).