ProBDNF induces neuronal apoptosis via activation of a receptor complex of p75NTR and sortilin

Abstract

Brain-derived neurotrophic factor (BDNF) is best characterized for critical roles in neuronal survival, differentiation, and synaptic modulation mediated by the TrkB receptor tyrosine kinase. Developmentally regulated death signaling by BDNF has also been demonstrated via activation of p75NTR. Because recent studies suggest that proNGF, the precursor form of NGF, is more active than mature NGF in inducing apoptosis after binding to p75NTR and a coreceptor, sortilin, we asked whether the precursor of BDNF (proBDNF) is also a proapoptotic ligand in the nervous system. proBDNF is secreted by cultured neurons, and recombinant proBDNF binds to sortilin. In sympathetic neurons coexpressing sortilin and p75NTR, we found that proBDNF is an apoptotic ligand that induces death at subnanomolar concentrations. In contrast, mature BDNF, but not proBDNF, is effective in inducing TrkB phosphorylation. proBDNF effects are dependent on cellular coexpression of both p75NTR and sortilin, because neurons deficient in p75NTR are resistant to proBDNF-induced apoptosis, and competitive antagonists of sortilin block sympathetic neuron death. Moreover, addition of preformed complexes of soluble sortilin and proBDNF failed to induce apoptosis of cells coexpressing both sortilin and p75NTR, suggesting that interaction of proBDNF with both receptors on the cell surface is required to initiate cell death. Together with our past findings, these data suggest that the neurotrophin family is capable of modulating diverse biological processes via differential processing of the proneurotrophins.

Figure 1.

proBDNF is released from cultured cortical neurons. A, Specificity of proBDNF antisera. Media from 293 cells infected with adeno-BDNF were incubated with proBDNF-specific IgY coupled to Sepharose (proBDNF IP) or comparably treated Sepharose in which IgY was not included in the coupling reaction (Control IP). Immunoprecipitated proteins were separated by SDS-PAGE and Western blotted with anti-mature BDNF. B, Conditioned media (40 ml from 1 × 10⁸ cells) and detergent lysates (2 mg; prepared in RIPA buffer) from cultured cortical neurons were immunoprecipitated with anti-proBDNF-coupled Sepharose beads. Immunoprecipitated proteins were separated by SDS-PAGE and Western blotted with anti-mature BDNF antiserum. The positions of proBDNF along with molecular weight markers (numbers in kilodaltons) are indicated. IP, Immunoprecipitation.

Figure 2.

Purification of proBDNF and mature BDNF. A, B, Recombinant adenovirus encoding BDNF-His (A) or proBDNF-His (B) was used to infect HEK 293 cells, and conditioned media were purified using Ni-chromatography. Lane 1, Conditioned media before chromatography. Lane 2, Column flow-through. Lane 3, Column wash. Lane 4, Column eluate. Lane 5, Column eluate subjected to silver stain analysis. C, Purification of proBDNF-His from media of baculovirus-infected insect cells. Lane 1, Conditioned media before chromatography. Lane 2, Column flow-through. Lane 3, Column wash. Lane 4, Column eluate. Lane 5, Column eluate subjected to silver stain analysis. D, ProBDNF-His column eluates (from lane 4, B) were reprobed with antibody specific for the pro-domain of BDNF. E, Medium from 293 cells expressing proBDNF-His was harvested and purified using Ni-chromatography. Purified proteins were incubated without or with N-glycanase (25 mU/ml) at 20°C for 12 h. Reactions were terminated by addition of SDS sample buffer and boiling. Proteins were separated by SDS-PAGE and Western blotted with anti-mature BDNF. When applicable, positions of proBDNF, mature BDNF, and their deglycosylated forms are indicated by arrows. WB, Western blot.

Figure 3.

Secreted proBDNF is dimeric. ProBDNF (non-His-tag) from recombinant adenovirus-infected cell media was purified using cation-exchange chromatography as described. Purified proBDNF was fractionated by Superdex 200 gel filtration in 25 mm Tris-Cl, pH 7.0, and 200 mm NaCl. The indicated fractions were analyzed by Western blotting with anti-BDNF antiserum. Top, Chromatogram of proBDNF elution profile. Native molecular weight standards are indicated by arrows. The peak of eluted proBDNF (arrow) has a calculated molecular weight of 68 kDa. Bottom, Western blot (WB) of eluates containing proBDNF.

Figure 4.

ProBDNF binds to sortilin. A, Surface plasmon resonance analysis of proBDNF binding to sortilin. Purified extracellular domain of sortilin was immobilized on a biosensor chip (120 fmol/mm²), followed by incubation with proBDNF purified from baculovirus-infected insect cell media at the indicated concentrations. The on and off rates were recorded, and the calculated K_d value is indicated. Inhibition of proBDNF (10 nm) binding to sortilin by 10 μm neurotensin (NT) is indicated, as are the response units obtained with diluent alone. B, 293 cells stably expressing proBDNF-His were infected with adenovirus (Ad) expressing GFP or soluble sortilin. At 2 d after infection, cell lysates were harvested and subjected to immunoprecipitation (IP) with sortilin antisera. Immunoprecipitated proteins were resolved by SDS-PAGE and Western blotted with anti-sortilin (top panels) or anti-mature BDNF (bottom panels) antisera. Cell lysates were resolved and Western blotted in parallel. WB, Western blot.

Figure 5.

Secreted sortilin reduces degradation of proBDNF. Conditioned media from 293 cells infected with adenovirus (Ad) expressing GFP or soluble sortilin were mixed (4 h at 4°C) with conditioned media from cells expressing proBDNF-His. These mixtures were then incubated with plasmin at the indicated concentration for 1 h, followed by SDS-PAGE and Western blot analysis using anti-sortilin (top panels) or anti-BDNF (bottom panels) antisera.

Figure 6.

Mature BDNF is more effective than proBDNF in eliciting TrkB activation. Secreted proBDNF-His or mature BDNF-His was purified by Ni-chromatography. A, TrkB-expressing PC12 cells were cultured in serum-free media for 6 h, followed by treatment with 1 nm mature BDNF, 1 nm proBDNF, or diluent control (None) as indicated for 10 min. Cell lysates were prepared and immunoprecipitated (IP) with anti-Trk antisera, followed by SDS-PAGE, and Western blotted (WB) with the indicated antisera. B, TrkB-expressing PC12 cells were treated, or not, with proBDNF-His, mature BDNF-His, or commercial mature BDNF in media containing 1% serum. Forty-eight hours later, the percentages of neurite-bearing cells were scored by an observer blinded to the treatment conditions. Error bars indicate the values obtained from two independent experiments.

Figure 7.

Sortilin and p75^NTR are required for the proapoptotic action of proBDNF. A, Cultured rat SCG neurons were incubated with either mature BDNF or proBDNF as indicated for 48 h. The cultures were then fixed and processed for immunohistochemistry with Tuj1 (red), followed by TUNEL analysis (green). Nuclei were counterstained with DAPI (blue). Scale bar, 100 μm. B, Replicate cultures of NGF-deprived rat SCG neurons were treated, or not, with NGF (10 ng/ml), mature BDNF (4 ng/ml), or proBDNF (4 ng/ml) in the presence or absence of 20 μm neurotensin as indicated. Forty-eight hours later, cells were processed for TUNEL analysis as in A. The percentage of TUNEL-positive neurons was scored under each culture condition. The numbers were then normalized to those neurons that were NGF deprived (varied between 10 and 20% in different experiments). Error bars indicate SEM from three independently conducted experiments. Statistical significance (p < 0.05) between each paired sample is indicated by asterisks. C, Sympathetic neurons from p75^NTR-null or wild-type (WT) mice were established as described previously (Bamji et al., 1998). Replicate cultures were treated as in B and processed for TUNEL analysis. Error bars indicate SEM from three independently conducted experiments. Statistical significance (p < 0.05) between the paired sample is indicated by asterisks.

Figure 8.

Effect of proBDNF and the proBDNF-soluble sortilin complex on neuronal apoptosis. Conditioned media from 293 cells stably expressing proBDNF-His were incubated with conditioned media from cells infected with adenovirus encoding GFP (lane 1) or soluble sortilin (lane 2) for 4 h at 4°C. ProBDNF and proBDNF-sortilin complexes were purified by Ni-chromatography. A, Eluates were resolved by SDS-PAGE and Western blotted with anti-sortilin (top) or anti-BDNF (bottom) antisera. The positions of soluble sortilin and proBDNF are indicated on the right (arrows). B, Rat SCG neurons were treated with the indicated ligands and processed for TUNEL analysis as described in Figure 7B. Error bars indicate SEM from three independently conducted experiments. When applicable, statistical significance between the indicated pairs of samples was presented.