Sortilin controls intracellular sorting of brain-derived neurotrophic factor to the regulated secretory pathway - PubMed (original) (raw)

Sortilin controls intracellular sorting of brain-derived neurotrophic factor to the regulated secretory pathway

Zhe-Yu Chen et al. J Neurosci. 2005.

Abstract

Brain-derived neurotrophic factor (BDNF), after activity-dependent secretion from neurons, modulates critical nervous system functions. Recently, a variant in the human bdnf gene, resulting in a valine to methionine substitution in the prodomain, has been shown to lead to defective regulated secretion from neurons and memory impairment. Here, we report a novel function for a Vps10p domain protein, sortilin, in controlling BDNF sorting to the regulated secretory pathway. Sortilin interacts specifically with BDNF in a region encompassing the methionine substitution and colocalizes with BDNF in secretory granules in neurons. A truncated form of sortilin causes BDNF missorting to the constitutive secretory pathway without affecting neurotrophin-4 (NT-4) secretion. In addition, sortilin small interfering RNA introduced into primary neurons also led to BDNF missorting from the regulated to the constitutive secretory pathway. Together, these data suggest a mechanism to understand the defect associated with variant BDNF and provide a framework, based on divergent presynaptic regulation of sorting to secretory pathways, to explain how two ligands for tropomyosin-related kinase B, BDNF and NT-4, can mediate diverse biological responses.

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Figures

Figure 1.

Figure 1.

Coimmunoprecipitation of sortilin with wild-type and variant BDNF. A, HEK 293 cells were cotransfected with cDNAs encoding sortilin and C-terminal BDNFVal-HA or BDNFMet-HA or empty vector (-). Cell lysates were immunoprecipitated with HA antibodies and immunoblotted with sortilin antibodies (top). Immunoprecipitation (IP) of BDNF-HA was confirmed by immunoblotting with HA antibodies (middle). Crude lysates were immunoblotted with sortilin antibodies and HA antibodies to confirm equivalent expression level (bottom). B, In vitro binding assay. GST-BDNFVal, GST-BDNFMet, or GST-NT-4 prodomain fusion proteins were incubated with lysates from HEK 293 cells overexpressing full-length sortilin. Bound sortilin was detected by immunoblotting with anti-sortilin antibody.

Figure 2.

Figure 2.

Mapping the interaction of sortilin with BDNF mutants. A, HEK 293 cells were cotransfected with sortilin and either full-length BDNF or NT-4 or truncation mutants. Lysates were immunoprecipitated with HA antibodies and immunoblotted with sortilin antibodies (top). Immunoprecipitation of BDNF-HA was confirmed by immunoblotting with HA antibodies (middle). Sortilin expression was verified with sortilin antibodies (bottom). B, Quantitation of Western blots in the top panel of A as a percentage of sortilin coimmunoprecipitation (Co-IP) by mutant neurotrophins compared with wild-type BDNF. Mean proportions ± SEM were determined from analysis of three independent experiments. C, Summary of BDNF and NT-4 mutants used in A and a summary of the sortilin interactions with the mutant BDNF and NT-4. IP, Immunoprecipitation; wt, wild type; m, mature.

Figure 3.

Figure 3.

Mapping the interaction of BDNF with sortilin mutants. A, Coimmunoprecipitation of tSort with BDNF. HEK 293 cells were cotransfected with cDNAs encoding a mutant truncated sortilin or full-length sortilin along with C-terminal-tagged BDNF-HA. Cell lysates were immunoprecipitated with HA antibodies and immunoblotted with sortilin antibodies (top). Immunoprecipitation of BDNF-HA was confirmed by immunoblotting with HA antibodies (middle). Crude lysates were immunoblotted with sortilin antibodies to confirm equivalent expression level (bottom). B, Endogenous association of BDNF with sortilin. P10 hippocampal and cortical lysates were subjected to immunoprecipitation with anti-BDNF antibodies or normal rabbit IgG and immunoblotted with sortilin antibodies (top). Immunoprecipitation of BDNF was confirmed by immunoblotting with BDNF antibodies (middle). Crude lysates were immunoblotted with sortilin antibodies to confirm equivalent protein loading (bottom). C, Constitutive secretion of BDNF in the absence and presence of truncated sortilin. HEK 293 cells stably expressing proBDNF were infected with recombinant adenovirus encoding GFP or tSort at multiplicity of infection of 1. Media (25 μg per lane) was then collected after 48 h after infection and immunoblotted with BDNF and sortilin antibodies. Wt, Wild type; IP, immunoprecipitation; Hippo, hippocampus; Con, control.

Figure 4.

Figure 4.

Subcellular colocalization of variant and wild-type BDNF with sortilin. A, Differentiated PC12 cells were transfected with N-terminal HA-tagged variant (Met) or wild-type BDNF (Val). Cells were fixed after 48 h and permeabilized. Colocalization of BDNF and sortilin was visualized by costaining with anti-HA and anti-sortilin antibodies. In addition, untransfected differentiated PC12 cells were fixed after 48 h and permeabilized. Colocalization of endogenous sortilin and SecII was visualized by costaining with anti-sortilin and anti-SecII antibodies. Representative epifluorescence images are shown. B, Hippocampal neurons were transfected with N-terminal HA-tagged variant (Met) or wild-type BDNF (Val) and Myc-tagged sortilin. Cells were fixed after 48 h and permeabilized. Colocalization of HA-BDNF and Myc-sortilin was visualized by confocal microscopy, as described in Materials and Methods, using epitope antibodies. All neurons were confirmed using MAP2 antibodies. In addition, untransfected neurons were fixed after 48 h and permeabilized. Colocalization of endogenous sortilin and SecII was visualized by costaining with anti-sortilin and anti-SecII antibodies. Representative epifluorescence images are shown. Scale bars, 20 μm.

Figure 5.

Figure 5.

Truncated sortilin alters intracellular BDNF targeting to secretory granules. A, Differentiated PC12 cells were cotransfected with full-length or truncated Myc-tagged sortilin along with N-terminal HA-tagged BDNFVal or BDNFMet. Cells were fixed after 48 h and permeabilized. Localization of sortilin (full-length, truncated), BDNF, and SecII was visualized by costaining with anti-HA, anti-Myc antibodies, and anti-SecII antibodies. Representative epifluorescence images are shown. B, Hippocampal neurons were cotransfected with truncated Myc-tagged sortilin along with N-terminal HA-tagged BDNF. Cells were fixed after 48 h and permeabilized. Localization of BDNF and SecII was visualized by costaining with anti-HA, anti-SecII antibodies, and anti-MAP2 antibodies. Localization of truncated sortilin and BDNF and SecII was visualized by costaining with anti-HA, anti-Myc antibodies, and anti-SecII antibodies. Representative epifluorescence images are shown. Scale bars, 20 μm.

Figure 6.

Figure 6.

Role of the BDNF prodomain on activity-dependent BDNF secretion. A, Lentiviral constructs containing differential regions of the BDNF prodomain (Box1, Box2, Box3, Box2+ 3) fused to GFP or GFP alone were expressed in PC12 cells transfected with N-terminal HA-BDNF. After 48 h, media were collected under depolarization secretion conditions, as described in Materials and Methods, and analyzed by ELISA. Results are presented as a mean ± SEM determined from six independent experiments (**p < 0.001; Student's t test). B, Differentiated PC12 cells were transfected with mutant forms of BDNF lacking certain regions of the prodomain (ΔBox1, ΔBox2′, ΔBox3, ΔBox2 + 3). After 48 h, media were collected under depolarization secretion conditions, as described in Materials and Methods, and analyzed by ELISA. Results are presented as a mean ± SEM determined from analysis of six independent experiments (**p < 0.001; Student's t test). WT, Wild type.

Figure 7.

Figure 7.

Truncated sortilin selectively alters activity-dependent BDNF secretion. A, Differentiated PC12 cells were transfected with NT-4 in the presence or absence of tSort. After 48 h, media were collected under depolarization and constitutive secretion conditions, as described in Materials and Methods, and analyzed by ELISA. Differentiated PC12 cells were transfected with BDNF in the presence or absence of full-length sortilin (Sort) or tSort. After 48 h, media were collected under depolarization (B) and constitutive secretion (C) conditions, as described in Materials and Methods, and analyzed by ELISA. All results are presented as a mean ± SEM determined from analysis of six independent experiments (*p < 0.01; **p < 0.001; Student's t test).

Figure 8.

Figure 8.

Truncated sortilin and sortilin siRNA selectively alter activity-dependent BDNF secretion. Hippocampo-cortical neurons were transfected with BDNF in the presence or absence of full-length sortilin (Sort), tSort, or sortilin siRNA (siSort). After 3 d, media were collected under depolarization (A) and constitutive secretion (B) conditions, as described in Materials and Methods, and analyzed by ELISA. All results are presented as a mean ± SEM determined from analysis of six independent experiments (*p < 0.05; **p < 0.01; Student's t test). C, Hippocampo-cortical neurons were transfected in the presence or absence of sortilin siRNA. After 3 d, cells lysates were immunoblotted for and sortilin and tubulin (loading control) as described in Materials and Methods.

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