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Originally published In Press as doi:10.1074/jbc.M300961200 on September 11, 2003

J. Biol. Chem., Vol. 278, Issue 48, 48129-48136, November 28, 2003
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Neurotrophin-4, Alone or Heterodimerized with Brain-derived Neurotrophic Factor, Is Sorted to the Constitutive Secretory Pathway*

Andrew P. Hibbert{ddagger}§, Stephen J. Morris{ddagger}||, Nabil G. Seidah**, and Richard A. Murphy{ddagger}{ddagger}{ddagger}

From the {ddagger}Centre for Neuronal Survival, Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada, the §Department of Developmental Biology, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada, and **Institut de Recherches Cliniques de Montréal, Montreal, Quebec H2W 1R7, Canada

Received for publication, January 29, 2003 , and in revised form, September 9, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Nerve growth factor and neurotrophin-3 (NT-3) are processed within the constitutive secretory pathway of neurons and neuroendocrine cells and are released continuously in an activity-independent fashion. In contrast, brain-derived neurotrophic factor (BDNF) is processed in the regulated secretory pathway, stored in vesicles, and released in response to neuronal activity, consistent with its role in modulating synaptic plasticity. In this study, we used vaccinia virus infection and transfection methods to monitor the processing and sorting of neurotrophin-4 (NT-4) in AtT-20 cells, which have been used as a model for the sorting of secretory proteins in neurons. Our data show that NT-4 is processed in the constitutive secretory pathway. The molecule is diffusely distributed within the cells and released, soon after being synthesized, in a manner that is not affected by cell depolarization. We further show that NT-4 and BDNF, when co-expressed, can form heterodimers that are constitutively released. In contrast, heterodimers of NT-3 and BDNF have been shown to be released through the regulated secretory pathway. Thus, NT-4, alone or when co-expressed with BDNF, is processed within and secreted by the constitutive secretory pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The neurotrophins nerve growth factor (NGF),1 brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4) (1) are all synthesized in pre-pro forms from which the signal peptide is cleaved in the endoplasmic reticulum to yield the propeptide. The N-terminal prodomain is removed in the trans-Golgi network or in immature secretory vesicles by proprotein convertases to yield the mature bioactive forms of the molecules (2–5). Neurotrophins are released and act as homodimers. In addition, bioactive neurotrophin heterodimers containing two single subunits of different neurotrophins are easily formed experimentally (6–9). There is no evidence, however, that heterodimers normally exist in the nervous system.

Previously, we and others have examined the post-cleavage fate of neurotrophins in neurons and neuroendocrine cells to distinguish whether neurotrophins are sorted into the constitutive or regulated secretory pathways. In the constitutive pathway, proteins are rapidly secreted following cleavage, whereas proteins in the regulated pathway are stored within secretory vesicles and released in an activity-dependent manner or in response to extracellular cues (see Ref. 10 for review).

Results show that BDNF is sorted to the regulated secretory pathway of both hippocampal neurons and AtT-20 cells (3, 11, 12), whereas NGF and NT-3 are sorted into the constitutive pathway (2, 3). Others have reported the regulated release of NGF and NT-3 (13–15), which may have resulted from high level expression in cultured cells (2, 3). Heterodimers of BDNF and NT-3 are also sorted into the regulated pathway, suggesting that BDNF in some way can exert a dominant influence over the sorting fate of a neurotrophin that is normally slated for continuous release (2).

NT-4 (also designated NT-5 or NT-4/5) was first identified in Xenopus and viper (16) and subsequently in mammals (17, 18). Like the other neurotrophins, NT-4 promotes the survival of peripheral (19, 20) and central (21) neurons. NT-4 also potentiates transmission at Xenopus neuromuscular junctions in culture (22) and may play a role in learning and long lasting long term potentiation (23, but see also Ref. 24). However, it is not known whether these effects are promoted directly, following activity-dependent secretion of NT-4, or whether NT-4 is released constitutively and so may only play a permissive role in modulating activity-dependent processes.

In this study, we examined the intracellular sorting of NT-4 in neuroendocrine AtT-20 cells, a common model in which to study the sorting of secretory proteins (10, 25, 26). Results show that NT-4 is sorted into the constitutive pathway. We further show that heterodimers of NT-4 and BDNF are constitutively secreted, unlike heterodimers of BDNF and NT-3, suggesting that coupling NT-4 to BDNF diverts BDNF from the regulated to the constitutive pathway.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—AtT-20 cells were maintained in DMEM (Biowhittaker, Walkersville, MD) containing 10% fetal bovine serum (Sigma), 1% glutamax (Invitrogen), and 0.4% gentamicin (Invitrogen) at 37 °C and 5% CO2.

Vaccinia Virus Infections—Purified vaccinia virus encoding rat pro-NT-4 was obtained essentially as described for PC1 and PC2 (27, 28). We used the full-length rat NT-4 coding region inserted at the BamHI (5') and HindIII (3') sites of the transfer vector PMJ601. The full-length cDNA of pro-NT-4 was generously provided by Regeneron Inc. For metabolic labeling studies, we plated 5 x 105 AtT-20 cells in each well of a 6-well plate (BD Biosciences) and allowed the cells to recover for 2 days. Each dish was then incubated for 1 h with 2.5 x 106 plaque-forming units vaccinia virus in 1.5 ml of phosphate-buffered saline (PBS) containing 0.01% bovine serum albumin and 1 mM magnesium chloride. The virus was removed, and the cells were allowed to recover for 8 h in supplemented DMEM. For immunostaining, 2 x 105 AtT-20 cells were plated per 60-mm diameter dish (BD Biosciences) and infected with 106 plaque-forming units in 2 ml/dish.

Transfections—Rat prepro-NT-4 sequence was extracted from pBluescript SK (Stratagene, La Jolla, CA) by PCR using primers KS (Stratagene), 5'-ATGGTGATGGTGATGCGATCCTCGGGCACGGCCTGTTCGGCTGAGGA-3' and 5'-TCTTAAGCTTGAATTCTACATCATCATCATGTGATGGTGATGGTGATGCGATCC-3'. The product, NT-4-RGS6His (RGS6His is a protein tag with the sequence RGSHHHHHH), was cloned into the EcoRI site of pCDNA3 (Invitrogen) to produce NT-4-RGS6His pCDNA3. pCDNA3 vector containing the sequence for BDNF-HA (BDNF-HA pCDNA3) was produced using a similar strategy. pCDNA3 and EGFP-N1 (Clontech) were used for some transfections.

Transfections with Superfect (Qiagen, Valencia, CA) were carried out using a modification of the manufacturer's instructions. For monitoring the release of proteins in response to depolarization, we plated 5 x 105 AtT-20 cells/well of a 6-well plate and allowed the cells to recover for 2 days. A total of 4 µg of plasmid DNA in Tris/EDTA buffer was mixed with 100 µl of DMEM with no supplements. For co-transfections, equal amounts of each plasmid were used. Superfect (13.3 µl) was mixed into the solution by vortexing for 5 s. The suspension was incubated at room temperature for 10 min before being mixed with 600 µl of complete medium, which was added immediately to the cells. After a 3-h incubation at 37 °C and 5% CO2, the mixture was removed and replaced with 1.5–2 ml of complete medium.

For immunostaining, the same procedures were followed with some modifications. 1.1 x 105 cells were plated into each chamber of a four-chambered slide (Nalge Nunc International, Naperville, IL). Complexes were formed between a total of 0.43 µg of DNA and 2.85 µl of Superfect in 12.8 µl of DMEM. After the incubation, 87.5 µl of complete medium was added to the mixture before complexes were added to the cells, and following transfection, the cells were maintained in 0.5 ml of DMEM.

In experiments in which the cells were transfected with two different vectors, it was assumed that any cell transfected with one vector would also express the other. This assumption is based on the nature of the Superfect-DNA complex. Each complex contains multiple plasmids. When two plasmids are used in equal amounts, the probability of all plasmids in a single complex being identical is very low. This was supported by our observations of cells co-transfected with pCDNA3 encoding BDNF along with EGFP-N1. Very few cells immunopositive for the neurotrophin failed to also express GFP.

Metabolic Labeling and Immunoprecipitation—Vaccinia virus-infected AtT-20 cells were washed twice and incubated for 1 h in Cys-Met-free DMEM (Biowhittaker) containing 5% fetal bovine serum, 1% glutamax, and 0.4% gentamicin. The cells were incubated for 1 h in 1 ml of the same medium containing 0.3 mCi 35S translabel (PerkinElmer Life Sciences) (70% methionine, 30% cysteine). Cells were washed twice and incubated with 1 ml of medium supplemented with excess non-radioactive cysteine/methionine for 0–8 h, and the media were saved. The cells were washed three times with PBS and then lysed in 1 ml of RIPA (150 mM NaCl, 1.0% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris-Cl, pH 8.0, with protease inhibitors). The lysate was centrifuged at 16,000 x g for 30 min, and the supernatant was collected.

Non-specific binding was reduced by incubating lysates and media with protein A-Sepharose beads (Amersham Biosciences) saturated with normal rabbit serum. To immunoprecipitate NT-4, we incubated the supernatants with 0.8 µg of anti-NT-4 (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at 4 °C with rotation. (In controls, non-immune serum replaced the primary antibody or 50 µl of the peptide against which the antibody was raised (Santa Cruz Biotechnology) was added along with the antibody.) Twenty µl of 50% protein A-Sepharose in RIPA was added to the samples and incubated for 1 h. Immunoprecipitates were washed four times with 1 ml of RIPA, boiled for 5 min in 50 µl of sample buffer, vortexed, and centrifuged.

The supernatants were analyzed by 13–22% SDS-PAGE. The samples were transferred to a nitrocellulose membrane (Osmonics, Westborough, MA), which was rinsed, dried, and exposed to a PhosphorImager screen (Amersham Biosciences).

Deglycosylation Experiments—Cells were lysed immediately following incubation with 35S translabel and immunoprecipitated as described above. After two washes with RIPA, the beads were treated as follows. For N-glycanase digestion, the beads were resuspended in 188 µlof0.2 M NaPO4, pH 7.6, 0.1% SDS, and 10 mM {beta}-mercaptoethanol and incubated at room temperature for 5 min. Twelve µl of 10% Nonidet P-40 was added and incubated for 5 min at room temperature. N-glycanase (4.5 µl, New England Biolabs, Beverly, MA) was added, and the beads were incubated at 37 °C for 18 h. Low salt wash buffer (900 µl, 2 mM EDTA, 0.5 mM dithiothreitol, 10 mM Tris, pH 7.5) was added, and the beads were centrifuged and washed twice more with low salt buffer followed by boiling in sample buffer and electrophoresis on SDS gels as described above. For endoglycosidase H digestion, the beads were washed once with incubation buffer (20 mM NaPO4, pH 6.0, 20 mM NaCl) and resuspended in 200 µl of incubation buffer. 5 µl of endoglycosidase H (Roche Applied Science, Laval QC) was added, and the beads were incubated for 18 h at 4 °C. The beads were washed three times with low salt buffer and analyzed as described above.

Immunostaining—Transfected cells were washed twice with PBS, incubated for 15 min in ice-cold 4% paraformaldehyde in PBS, washed three times for 5 min each with cold PBS, incubated for 5 min in ice-cold 0.5% Triton X-100 in PBS, rinsed once with PBS, and blocked for at least 2 h in blocking solution (6% normal goat serum plus 0.5% bovine serum albumin in PBS).

The blocking solution was removed, and the cells were incubated with the primary antibodies in 50% blocking solution overnight at 4 °C. Anti-NT-4 (Chemicon, Temecula, CA) and anti-HA (Covance, Richmond, CA) were diluted 1:500, anti-EEA1 (BD Biosciences) was diluted 1:2500, and anti-GM130 (BD Biosciences) was diluted 1:250. The cells were washed three times with 1 ml of PBS for 5 min and incubated in 1:500 anti-rabbit Cy-3 (Jackson Immunoresearch Laboratories, West Grove, PA) or 1:500 anti-mouse Cy-3 along with 1:500 Alexa 488-conjugated anti-rabbit (Molecular probes, Eugene, OR) for the co-staining experiments, in 50% blocking solution for 1 h in the dark at room temperature. The cells were washed and mounted in Immunon (Shandon, Pittsburgh, PA). Epifluorescence was visualized using a Zeiss LSM5 Pascal confocal microscope. Images were prepared for publication using Adobe PhotoShop. In some cases, {gamma} adjustment was used to emphasize the signals.

Neurotrophin Release—Neurotrophin release from cells was stimulated and measured using a modification of a method described previously (11). Briefly, cells were washed 3 x 15 min at 37 °C, 5% CO2 with a control solution (127 mM NaCl, 5 mM KCl, 0.33 mM Na2HPO4, 0.44 mM KH2PO4, 4.2 mM NaHCO3, 5.2 mM CaCl2, 5.6 mM glucose, 10 mM Hepes, pH 7.4, 1 mg/ml bovine serum albumin) followed by a 30-min incubation with 1 ml of the same solution. The solution from this incubation was saved, and the cells were incubated for 30 min with 1 ml of a depolarizing solution (the same as for the control solution except with 77 mM NaCl and 55 mM KCl). Cells were washed three times with PBS, lysed in 1 ml of RIPA. The lysate was centrifuged for 30 min at 16,000 x g, and the supernatant was saved.

To detect the RGS6 His-tagged NT-4, we brought the samples to 20 mM imidazole, 10% fetal bovine serum, and incubated them with 20 µl of Ni-NTA-agarose beads (Qiagen) overnight at 4 °C with rotation. The beads were washed three times with 50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0, before being boiled in sample buffer, vortexed, centrifuged, and separated by 15% SDS-PAGE. The samples were transferred to nitrocellulose (Osmonics) (400 mA for 90 min), blocked for 1 h in 5% milk in Tris-buffered saline with Tween, and incubated overnight at 4 °C in 1:1000 or 1:250 anti-RGS6 His (Qiagen) in 5% milk. The blot was washed 3 x 15 min in Tris-buffered saline with Tween, incubated for 1 h at room temperature in 1:1000 anti-mouse peroxidase (Jackson Immunoresearch Laboratories), washed three times as before, and visualized using ECL (PerkinElmer Life Sciences). HA-tagged BDNF was immunoprecipitated using 1 µl of rabbit anti-HA (Covance) and protein A-Sepharose beads. Immunoblotting was performed with 1:1000 mouse anti-HA (Covance).

To specifically detect NT-4-RGS6 His/BDNF-HA heterodimers, samples were incubated with Ni-NTA-agarose beads, and the beads were washed as above. Following a fourth 5-min wash, the NT-4 RGS6 His-containing dimers were incubated in 50 µl of 50 mM NaH2PO4, 300 mM NaCl, 250 mM imidazole, pH 8.0, for 10 min, vortexed, and centrifuged, and the supernatant was analyzed by 15% SDS-PAGE. Immunodetection of the BDNF-HA was done using 1:1000 mouse anti-HA (Covance).

Quantitative Analysis—Exposed films were digitized in TIFF format and analyzed using Image J 1.29 (National Institutes of Health, Bethesda, MD). Following background subtraction, each band density, along with a background control, was quantified. For each analysis, the selection area was kept constant. For each band, the background was subtracted, and ratios were calculated. The data were statistically analyzed by two-way analysis of variance followed by Student Newman Keuls post-hoc test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Synthesis, Processing, and Glycosylation of Pro-NT-4 in Virally Transduced AtT-20 Cells—To examine the processing and sorting of NT-4, we infected AtT-20 cells with recombinant vaccinia virus encoding rat prepro-NT-4. The protein produced by these cells was metabolically labeled and immunoprecipitated with an antibody that recognizes an epitope in the C-terminal region of the mature form of NT-4. Specific bands corresponding to pro-NT-4 and mature NT-4 were observed (Fig. 1A) that were not immunoprecipitated by non-immune rabbit serum or when excess competing peptide was added along with the antibody. The protein evident on the gel with a lower apparent molecular weight than the precursor was induced in response to wild-type virus infection (data not shown) and therefore was non-specific.



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  		FIG. 1.
Immunoprecipitation of metabolically labeled NT-4. As shown in A, to test antibody specificity, we collected cell lysates immediately following a 1-h incubation of pro-NT-4 expressing AtT-20 cells with [35S]cysteine/methionine (0 h lysate) or media conditioned by similar cells over 8 h (8 h media). Samples were immunoprecipitated with an antibody to NT-4 alone, the antibody in the presence of excess competing peptide, or non-immune rabbit serum (NRS). Antibody complexes were collected and separated by SDS-PAGE. Radiolabeled protein was visualized using a phosphorimaging screen. As shown in B, pro-NT-4 is glycosylated. Metabolically labeled cell lysates were treated for 18 h with either N-glycanase or endoglycosidase H (Endo-H) and separated by SDS-PAGE.

 
The molecular mass predicted from the primary amino acid sequence of pro-NT-4 is 20 kDa. However, in our experiments, pro-NT-4 has an apparent molecular mass of ~28 kDa, suggesting that the precursor is glycosylated, as has been demonstrated with NGF (4) and BDNF (29). To test for glycosylation, we digested the immunoprecipitated lysate with either N-glycanase or endoglycosidase H. Removal of N-linked carbohydrate residues with N-glycanase reduced the apparent molecular mass of the NT-4 precursor to ~23 kDa. (Fig. 1B). Treatment with endoglycosidase H, which removes high mannose sugar moieties, also reduced the apparent molecular mass of the precursor. In both cases, the non-specific band seen in Fig. 1A is also detected. Absence of an NT-4 endoglycosidase H-resistant band suggests that pro-NT-4 is present in the endoplasmic reticulum and rapidly cleaved upon entering the trans-Golgi network, as reported previously for other neurotrophins (4, 5). Alternatively, pro-NT-4 may remain endoglycosidase H-sensitive even upon entering the trans-Golgi network, as has been reported for the convertases PC1 and PC2 (30).

Kinetics of Processing and Release of NT-4—To examine the processing and release of NT-4, we metabolically labeled virally infected AtT-20 cells and monitored the time course of processing and release of NT-4 by immunoprecipitation and SDS-PAGE. Fig. 2 shows that the precursor is rapidly cleaved to yield mature NT-4, with almost no precursor evident in cell extracts after 2 h. At the same time, significant amounts of mature NT-4 accumulate in the conditioned media. Processed NT-4 was not detected in the lysate at any time, indicating that, rather than being stored intracellularly, it is primarily released rapidly after being cleaved. Released NT-4 begins to disappear with chase times longer than 2 h, probably due to proteolytic degradation in the media and/or cellular endocytosis followed by lysosomal degradation. The lack of retention of NT-4 within the cells and its rapid release is characteristic of proteins secreted from the constitutive secretory pathway and was similar to that of NGF (3) and NT-3 (2) but not BDNF (2, 3). We did not detect secretion of pro-NT-4 under these conditions. Previous studies have failed to detect secretion of pro-NGF or pro-NT-3 by identical assays, although significant amounts of pro-BDNF were secreted (2, 3).



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  		FIG. 2.
Pulse-chase metabolic labeling of pro-NT-4. AtT-20 cells were infected with recombinant vaccinia virus encoding rat pro-NT-4. Cells were incubated for 1 h with [35S]cysteine/methionine and chased for various times with excess non-radioactive amino acids. Conditioned media (M) and cell lysates (L) were collected. Samples were immunoprecipitated with anti-NT-4, separated by SDS-PAGE, and radiolabeled protein visualized using a PhosphorImager screen.

 
NT-4 Is Constitutively Secreted—The secretion kinetics we observed suggested that NT-4 is in the constitutive secretory pathway rather than the regulated secretory pathway. To test this idea further, we asked whether cell depolarization would stimulate NT-4 release, which would only occur if NT-4 were in the regulated pathway. AtT-20 cells were transfected with NT-4-RGS6His plasmid, and conditioned medium was collected over 2 days following transfection. The cells were exposed to non-depolarizing solution and depolarized for 30 min, and finally, the cells were lysed. Mature NT-4-RGS6His was evident in all four samples, with no difference between the amounts released into the non-depolarizing and depolarizing media (Fig. 3A). Thus, the release of NT-4 was not influenced by depolarization. We were able to detect a significant amount of pro-NT-4 in the media conditioned over 2 days, but not in either of the test solutions, each conditioned for only 30 min (Fig. 3A). In contrast, Fig. 3B shows that the depolarizing solution stimulated the release of BDNF, which is known to be in the regulated secretory pathway, ~2-fold (11). In agreement with previous data (3) we also observed release of pro-BDNF into the media, although pro-BDNF was detected at far lower levels than the mature protein. In many, but not all cases, pro-BDNF was also detected in the 30-min test solutions. Pro-BDNF release was independent of activity (see Fig. 5, C and D). Our methods for detecting NT-4-RGS6His or BDNF-HA are specific in that no signal was detected from EGFP-N1 transfected cells by either method (Fig. 3).



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  		FIG. 3.
NT-4 release is not stimulated by depolarization. In A, AtT-20 cells were transfected with NT-4-RGS6His pCDNA3 or EGFP-N1. Conditioned medium was collected (M), and the cells were incubated consecutively in non-depolarizing (ND) and then depolarizing, high potassium (D) solutions for 30 min each, following which the cells were lysed (L). NT-4-RGS6His was collected on Ni-NTA beads and subjected to Western analysis using an antibody to the RGS-6His tag. In B, cells were transfected with BDNF-HA pCDNA3 or EGFP-N1. Samples were collected as above and immunoprecipitated with anti-HA polyclonal antibodies and then analyzed by Western blot using an anti-HA monoclonal antibody.

 



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  		FIG. 5.
NT-4/BDNF heterodimers form in co-transfected cells and are constitutively secreted. In A, conditioned media were collected over 2 days from AtT-20 cells co-transfected with NT-4-RGS6His pCDNA3, BDNF-HA pCDNA3, or pCDNA3 with no insert, as indicated. Neurotrophins were collected on Ni-NTA beads and separated on SDS-PAGE. BDNF-HA was detected with an anti HA monoclonal antibody. In B, conditioned media were collected as for panel A. The media used in lanes 1 and 3 from panel A were mixed and incubated at 37 °C for 24 h. The mixture, along with medium from cells co-expressing both NT-4-RGS6His and BDNF-HA (as in panel A, lane 2), also incubated at 37 °C as a positive control, was then precipitated with Ni-NTA beads and analyzed by Western blot using an anti-HA monoclonal antibody. In C, non-depolarizing physiological buffer (ND) and depolarizing, high potassium solution (D) were conditioned consecutively for 30 min each by transfected AtT-20 cells. Left panel, conditioned solutions from transfected cells were precipitated (P) with Ni-NTA beads followed by SDS-PAGE and immunodetection using anti-RGS6His. Right panel, conditioned solutions from transfected cells were immunoprecipitated (IP) and detected following SDS-PAGE by different HA antibodies. WB, Western blot. In D, secretion of BDNF, pro-BDNF, or NT-4 was expressed as the ratio of neurotrophin appearing in the depolarizing solution when compared with release under control conditions. Solid bars indicate release of the neurotrophin expressed alone, and open bars indicate release of the neurotrophin from cells co-expressing pro-NT-4 and pro-BDNF. The relative release was compared by analysis of variance (significant difference between ligands, p < 0.001, no significant effect of co-expression with other neurotrophin (p = 580), no significant interaction (p = 0.629)) followed by Student-Newman-Keuls post-hoc tests. ** indicates a significant increase (p < 0.001) in BDNF-HA release when compared with either other ligand. Error bars are S.E. In E, AtT-20 cells were transfected with NT-4-RGS6His pCDNA3, BDNF-HA pCDNA3, or pCDNA3 with no insert in pairs as indicated. Cells were exposed to non-depolarizing media (-KCl) and then depolarizing media (+KCl). NT-4-RGS6His-containing dimers were collected on Ni-NTA beads followed by anti-HA Western analysis to reveal co-precipitated BDNF-HA.

 
Intracellular Localization of NT-4—We turned to immunocytochemistry to examine the distribution of NT-4 in AtT-20 cells. Characteristically, proteins in the regulated secretory pathway of AtT-20 cells accumulate in the cell processes in small punctate structures corresponding to secretory granules. Conversely, proteins in the constitutive secretory pathways are diffusely distributed and generally do not accumulate at the tips of cell processes (2, 3, 25, 31).

In cells transfected with NT-4-RGS6His pCDNA3, NT-4 was diffusely distributed and did not accumulate at the tips of cell processes (Fig. 4). Similar results were obtained using vaccinia virus expressing pro-NT-4 (data not shown). The perinuclear signal corresponds to NT-4 in the Golgi apparatus, as shown by its co-localization with GM130, a marker for the Golgi apparatus (Fig. 4, A–C). This co-localization was not due to bleed-through between the Alexa 488 and Cy-3 channels or non-specific antibody interactions, as no co-localization was seen with the endosomal marker EEA1 (Fig. 4, D–F). Immunostaining was specific in that no signal was evident following transfection with pCDNA3 (data not shown). The diffuse distribution of NT-4 within the cell and its lack of accumulation in cell processes agree with our biochemical evidence suggesting that NT-4 is secreted via the constitutive secretory pathway. Consistent with previous reports (2, 3), BDNF-HA localized to puncta and accumulated in cell processes in BDNF-HA pCDNA3/pCDNA3 co-transfected cells (see Fig. 6B). In some experiments we observed, in a minority of cells, NT-4 immunostained with a distribution similar to BDNF. This might be due to those cells expressing NT-4 at a particularly high level, directing some protein to the regulated pathway, as has been reported for NGF and NT-3 (2, 3).



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  		FIG. 4.
NT-4 is diffusely distributed in AtT-20 cells. AtT-20 cells were transfected with NT-4 RGS6His pCDNA3 and allowed to recover for 2 days. The cells were immunostained using an antibody to NT-4 and an Alexa 488-conjugated secondary along with anti-GM130 (A–C) or anti-EEA1 (D–F) and Cy-3-conjugated goat anti-mouse. Fluorescence from Alexa 488 (A and D) and Cy-3 (B and E) is shown along with overlays of the two channels with the transmitted signal to indicate cell morphology (C and F).

 



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  		FIG. 6.
NT-4 distribution is not affected by co-expression with BDNF. In A, AtT-20 cells were transfected with NT-4 RGS6His pCDNA3 and BDNF-HA pCDNA3 and allowed to recover for 2 days. The cells were immunostained using an antibody to NT-4 and an Alexa 488-conjugated secondary along with anti-HA and Cy-3-conjugated goat anti-mouse. Fluorescence from Cy-3 and Alexa 488 is shown overlaid with the transmitted signal to indicate cell morphology. Arrowheads show HA immunoreactive puncta in the cell process. The region indicated by the arrowheads is magnified in the insets, which show the Cy-3 and Alexa 488 channels separately. In B and C, AtT-20 cells were transfected with BDNF-HA pCDNA3 or NT-4-RGS6his pCDNA3, along with control vector, and immunostained as above. The overlays of the Cy-3, Alexa 488, and transmitted signals are shown, along with insets showing the HA or NT-4 specific signals.

 
NT-4/BDNF Heterodimers Are Formed within Co-transfected Cells—Neurotrophins are normally released as homodimers, but co-expression of different members of the neurotrophin family can result in the formation of bioactive heterodimers (7, 32). Previously, we reported that co-expression of NT-3 with BDNF results in secretion of BDNF/NT-3 heterodimers and that heterodimerizing NT-3 with BDNF redirects NT-3 from the constitutive to the regulated secretory pathway (2).

To examine the fate of NT-4 heterodimerized with BDNF, we co-transfected cells with NT-4-RGS6His pCDNA3 and BDNF-HA pCDNA3, precipitated the NT-4-RGS6His-containing dimers with Ni-NTA beads (by virtue of the 6-His tag), and eluted the dimers with imidazole. The recovered material was then denatured, separated by SDS-PAGE, transferred to nitrocellulose, and probed with an antibody to HA to detect BDNF-HA. BDNF-HA was co-precipitated from media conditioned by NT-4-RGS6His pCDNA3 and BDNF-HA pCDNA3 co-transfected cells, indicating the presence of stable NT-4/BDNF heterodimers (Fig. 5A, lane 2). The HA antibody did not cross-react with proteins precipitated from cells transfected with NT-4-RGS6His (Fig. 5A, lane 1), and BDNF-HA was not precipitated from cells not also expressing NT-4-RGS6His (Fig. 5A, lane 3). All of the proteins were expressed as expected, as shown by precipitation and Western blot analysis, as in Fig. 3, A and B (data not shown).

To rule out the possibility that the heterodimers we detected formed extracellularly in the conditioned medium as a result of the non-specific aggregation of homodimers, we combined medium from cells transfected with NT-4-RGS6His pCDNA3 or BDNF-HA pCDNA3 alone and incubated it overnight at 37 °C. We were unable to co-immunoprecipitate NT-4-RGS6His and BDNF-HA from this mixture (Fig. 5B), indicating that the NT-4/BDNF heterodimers form intracellularly.

NT-4/BDNF Heterodimers Are Sorted to the Constitutive Secretory Pathway—Examination of NT-4-RGS6His release revealed that that co-expressing NT-4 with BDNF did not result in increased release of NT-4 from cells exposed to depolarizing conditions over control conditions (Fig. 5, C and D). This indicates that NT-4 remains in the constitutive pathway under conditions where heterodimers of the two neurotrophins form. Detectable levels of NT-4 were reduced in cells also expressing BDNF, which may reflect reduced binding of the heterodimers to the nickel beads or increased NT-4 degradation following the co-transfection. Such an effect has been reported previously in a similar system (7). In co-transfected cells, we were not able to detect a decrease in the depolarization-induced secretion of BDNF (Fig. 5, C and D) or an increase in its basal release (p = 0.22 by z-test). The unregulated release of pro-BDNF was not altered by co-transfection (Fig. 5, C and D).

To directly examine the secretion of the heterodimers, we specifically precipitated NT-4-RGS6His-containing dimers from the conditioned non-depolarizing and depolarizing solutions and analyzed the precipitates for HA immunoreactivity on Western blots. As discussed above, this method allows us to selectively detect NT-4-RGS6His/BDNF-HA heterodimers. Similar amounts of BDNF-HA were detected in both precipitates (Fig. 5E). This indicates that depolarization did not substantially increase the release of the heterodimers, consistent with constitutive secretion of the NT-4/BDNF heterodimers.

We next examined the intracellular distribution of NT-4 immunoreactivity in cells transfected with NT-4-RGS6His pCDNA3 along with pCDNA3 coding for pro-BDNF-HA. We were not able to detect punctate staining of NT-4 in cells also expressing BDNF (Fig. 6A), indicating that NT-4 remains in the constitutive pathway in the presence of BDNF. In some cases, small amounts of NT-4 were detected, which might show some co-localization with BDNF in the processes. However, such signals were weak and cannot be distinguished from the granular background staining. No NT-4-specific signal was seen in cells transfected with BDNF-HA pCDNA3 alone (Fig. 6B). Equally, no cross-reactivity of the HA antibody was seen with cells transfected with NT-4-RGS6His with control plasmid (Fig. 6C). In cells expressing NT4-RGS6His, we were able to detect punctate BDNF-HA staining (Fig. 6A) and accumulation of BDNF in cell processes. BDNF was similarly distributed following transfection with BDNF-HA pCDNA3 alone (Fig. 6B), indicating that the regulated secretory pathway is intact in the co-transfected cells. Therefore, in contrast to NT-3/BDNF heterodimers, which are sorted into the regulated secretory pathway (2), NT-4/BDNF heterodimers are sorted to the constitutive secretory pathway.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
AtT-20 cells contain both the regulated and constitutive secretory pathways and have been used extensively to study protein processing and sorting (25, 26, 33–36). Sorting of BDNF, NGF, and NT-3 in AtT-20 cells is identical to that observed in hippocampal neurons in culture (2, 3), suggesting that AtT-20 cells are a good model for studying the processing of secretory proteins in neurons.

Proteins in the regulated secretory pathway can be identified by three distinct criteria (10). In contrast to proteins in the constitutive pathway, they are stored and accumulate within cells, they are packaged in secretory granules that appear punctate by immunocytochemistry, and their release is induced by secretagogues. Using these criteria, we have shown previously that BDNF is processed and released by the regulated secretory pathway in AtT-20 cells and hippocampal neurons. In contrast, NGF and NT-3 are sorted to the constitutive secretory pathway (2, 3). Others have shown that BDNF, NGF, and NT-3 can all be released in a regulated manner (13–15, 37). However, regulated release of NGF and NT-3 occurs only when the proteins are expressed experimentally at high levels; overexpression can result in these proteins being rerouted from the constitutive to the regulated secretory pathway (2, 3).

In the present study, we show that NT-4 is released via the constitutive secretory pathway of AtT-20 cells. Metabolically labeled NT-4 precursor (pro-NT-4) is processed rapidly to generate the mature form of NT-4, which is quickly released into conditioned media. As a result, scant amounts of radiolabeled pro- or mature NT-4 remain in the cells after a 2-h chase. Also, the cellular distribution of NT-4 is diffuse and shows no punctate staining or accumulation at the tips of cell processes, the hallmarks of the regulated secretory pathway. Finally, depolarizing the cells for 30 min did not enhance the release of NT-4 when compared with cells exposed to a non-depolarizing control medium. Taken together, all three lines of evidence suggest strongly that NT-4 is processed in the constitutive secretory pathway.

One previous study that examined the release of NT-4 concluded that NT-4 release from PC12 cells could be increased by depolarization or exogenous NGF (14). These results could have occurred as a result of NT-4 being overexpressed, as has been reported for NGF and NT-3 (2, 3), although we are not able to compare the levels of NT-4 expression per cell in our study with those reported by Kruttgen et al. (14). Alternatively, neurotrophin sorting may differ between AtT-20 and PC12 cells. PC12 cells express both Trk and p75 neurotrophin receptors and are able to internalize neurotrophins (38, 39). To our knowledge, neither receptor has been found expressed in AtT-20 cells. The regulated release of neurotrophins from PC12 cells may reflect release of recycled rather than freshly produced protein (40).

In our study, two lines of evidence show that we are monitoring the fate of newly synthesized rather than recycled NT-4. By metabolic labeling, which identifies proteins produced during a specific time period, we show that mature NT-4 is undetectable in cell lysates, even 6 h after most labeled NT-4 has been released into the media, a finding that argues against intracellular storage of recycled NT-4. Also, NT-4 did not colocalize with the endosomal marker EEA1, arguing against internalization of NT-4 to the early endosomes.

There exists in the prodomain of pro-NT-4 a predicted N-linked glycosylation site (NX(T/S)) at position 75, which studies with N-glycanase digestion suggest is occupied. Similar glycosylation sites are occupied in the prodomains of pro-NGF, pro-NT-3, and pro-BDNF (4, 5, 29). Glycosylation of pro-NT-4 may be necessary for proper folding of the precursor or for maintaining its stability (29).

Co-expression of pro-BDNF and pro-NT-4 leads to the formation and secretion of heterodimers through the constitutive secretory pathway. By specifically detecting BDNF from heterodimers in the control and depolarizing solutions, we have shown that NT-4/BDNF heterodimer secretion is activity-independent. This conclusion is supported by our observations that NT-4, co-expressed with BDNF, is absent from BDNF-containing punctate structures and is not released in response to elevated concentrations of potassium. If the heterodimers were in the regulated pathway, we would expect even a small amount of NT-4 entering the secretory granules to accumulate in those structures over hours or days. It would therefore be easy to detect NT-4 by immunostaining or by following its release after depolarization.

We might expect that NT-4, by forming heterodimers with BDNF, is redirecting some BDNF, which would otherwise enter the regulated pathway. However, we did not detect a change in the secretion characteristics of BDNF when co-expressed with NT-4. This might indicate that BDNF sorting is unaffected by NT-4. It is possible that some BDNF is in the constitutive pathway and, by some mechanism, that that BDNF preferentially forms heterodimers with NT-4. Alternatively, our failure to detect a change may be a consequence of the technical limitations of our assays. The assays used in this study provide a powerful test of the hypothesis that a protein is found in the regulated secretory pathway. However, they do not allow quantitation of what proportion of the protein is in each pathway nor provide sensitive assays for a change in BDNF secretion. The amounts of BDNF released in response to depolarization and found in a punctate distribution are only indirectly dependent on the proportion sorted into the regulated pathway in the Golgi. Their storage and degradation is also subject to regulation. In addition, basal release of BDNF could be attributed to regulated release as a result of the spontaneous bursting action potentials that have been measured in resting AtT-20 cells (41) as well as constitutive secretion. This regulated secretion will partially mask any change in constitutive release. Consequently, none of these assays are sufficiently sensitive to allow us to rule out a small change in BDNF sorting upon co-expression with NT-4.

We were able to show directly that some BDNF, associated with NT-4, is released by the constitutive pathway following co-transfection. Heterodimers of NT-3 and BDNF are processed in the regulated secretory pathway (2). Understanding how NT-3/BDNF and NT-4/BDNF heterodimers are differentially sorted will require a detailed understanding of how the pro-neurotrophin subunits interact. One possibility is that pro-NT-4 may mask putative sorting signals in pro-BDNF that are not blocked by pro-NT-3. Alternatively, pro-NT-4 may contain a dominant sorting signal that directs it to the constitutive or constitutive-like secretory pathways, although to our knowledge, such a signal has never been reported. Our failure to detect a change in BDNF secretion in cells also expressing NT-4 raises a third possibility. The heterodimers may form within the constitutive secretory pathway, presumably in a post-Golgi compartment. Such a mechanism would be surprising as we are not aware of any examples of subunits of secreted proteins combining downstream of the Golgi apparatus. Indeed, another neurotrophin heterodimer, comprising NT-3 and BDNF, forms prior to the sorting decision in the Golgi apparatus, allowing NT-3 to be directed into the regulated pathway (2). We still do not know whether neurotrophin heterodimers form in vivo, but if they do, our work suggests that BDNF/NT-4 heterodimers will act in a very similar way to NT-4 homodimers.

Important recent reports have shown that pro-NGF is able to bind to the p75 neurotrophin receptor (NTR) with ~5-fold higher affinity than mature NGF (42) and that pro-NGF is much more effective than the cleaved protein in causing apoptosis of oligodendrocytes (43). This raises the possibility that pro-NGF and perhaps other pro-forms of the neurotrophins act as p75-NTR ligands in vivo. Some studies have detected pro-neurotrophins in tissue homogenates (42–44). As yet, however, it is not known whether pro-neurotrophins are secreted from cells in vivo, which would be required for the proteins to activate p75-NTR on neurotrophin-responsive cells.

A small amount of pro-NT-4 was detected in media conditioned for 2 days by transfected AtT-20 cells. Since we do not know whether the antibody used in our Western blot analysis detects pro-NT-4 and mature NT-4 with equal affinity, we cannot accurately compare the relative amounts of processed and unprocessed NT-4 released in this experiment. Our pulse-chase studies, in which we can directly compare the amounts of precursor produced and secreted, show that the proportion of pro-NT-4 that is secreted uncleaved from vaccinia virus-infected cells is extremely low, as we have seen previously for pro-NT-3 and pro-NGF. Using similar methods, we have not been able to detect secretion of pro-NGF (3) or pro-NT-3 (2). In contrast, intact pro-BDNF is released into medium by the constitutive secretory pathway from both transfected and vaccinia virus-infected cells (Ref. 3 and this study). However, we have no evidence that pro-BDNF is released intact in vivo.

It should be noted that significant amounts of unprocessed pro-BDNF and pro-NGF, but not pro-NT3, have been detected in medium conditioned by Cos cells, which contain only the constitutive secretory pathway (2, 13). Secretion of pro-NGF has also been reported from several cell lines following vaccinia virus infection (37). However, we cannot say whether constitutive secretion of the precursor is an artifact of overexpression in cultured cells or an accurate reflection of precursor release in vivo.

We have found that NT-4 is released constitutively, whereas BDNF is released by the regulated secretory pathway (3, 11, 45, 46). Therefore, two ligands that bind the same receptors, Trk B and the p75 neurotrophin receptor, are released by different mechanisms. This difference could explain differences in the effects of these ligands in vivo. For example, it has been shown that NT-4, when expressed under the BDNF locus, is unable to completely rescue the BDNF knockout phenotype (47). Although there may be differences in how NT-4 and BDNF signal through TrkB, differences in their modes of secretion may also play a role. Recent data support this hypothesis. It has been shown that a naturally occurring point mutant in the prodomain of BDNF leads to reduced activity-dependent secretion of BDNF and impairments in some memory tasks (12). Constitutive secretion of NT-4 may allow it to perform tasks distinct from BDNF, even within the same neuronal system.


   FOOTNOTES
 
* This work was supported by grants from the Canadian Institute of Health Research (to R. A. M. and to N. G. S.). 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

Supported by a scholarship from the McGill University (Canada) Trust, UK, and McGill University Faculty of Medicine. Back

|| Present address: Head of Cellular Screening, Aegera Therapeutics, 810 Chemin du Golf, Ile des Soeurs, Quebec, Canada. Back

{ddagger}{ddagger} To whom correspondence should be addressed: The Salk Institute for Biological Sciences, Office of the President, 10010 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-453-2480; Fax: 858-546-0838; E-mail: murphy{at}salk.edu.

1 The abbreviations used are: NGF, nerve growth factor; pro-NGF, the precursor to NGF; BDNF, brain-derived neurotrophic factor; pro-BDNF, the precursor to BDNF; EEA1, early endosome antigen 1; GM130, Golgi matrix protein of 130 kDa; HA, hemagglutinin; Ni-NTA, nickel-nitrilotriacetic acid; NT-3, neurotrophin-3; NT-4, neurotrophin-4; pro-NT-3, the precursor to NT-3; pro-NT-4, the precursor to NT-4; NTR, neurotrophin receptor; DMEM, Dulbecco's modified Eagle's medium; GFP, green fluorescent protein; EGFP, enhanced GFP; PBS, phosphate-buffered saline. Back


   ACKNOWLEDGMENTS
 
We would like to thank Drs. D. Kaplan and F. Miller for the use of equipment and reagents and F. Barnabe-Heider for technical assistance. We are also grateful to Drs. D. Kaplan and F. Miller and F. Barnabe-Heider for critical reading of the manuscript.



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 ABSTRACT
 INTRODUCTION
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 RESULTS
 DISCUSSION
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