Retrolinkin cooperates with endophilin A1 to mediate BDNF-TrkB early endocytic trafficking and signaling from early endosomes

Abstract

Brain-derived neurotrophic factor (BDNF) binds to its cell surface receptor TrkB to regulate differentiation, development, synaptic plasticity, and functional maintenance of neuronal cells. Binding of BDNF triggers TrkB dimerization and autophosphorylation, which provides docking sites for adaptor proteins to recruit and activate downstream signaling molecules. The molecular mechanisms underlying BDNF-TrkB endocytic trafficking crucial for spatiotemporal control of signaling pathways remain to be elucidated. Here we show that retrolinkin, a transmembrane protein, interacts with endophilin A1 and mediates BDNF-activated TrkB (pTrk) trafficking and signaling in CNS neurons. We find that activated TrkB colocalizes and interacts with the early endosome marker APPL1. Both retrolinkin and endophilin A1 are required for BDNF-induced dendrite development and acute extracellular signal-regulated kinase activation from early endosomes. Suppression of retrolinkin expression not only blocks BDNF-triggered TrkB internalization, but also prevents recruitment of endophilin A1 to pTrk vesicles trafficking through APPL1-positive endosomes. These findings reveal a novel mechanism for BDNF-TrkB to regulate signaling both in time and space through a specific membrane trafficking pathway.

FIGURE 1:

Retrolinkin is preferentially expressed in dendrites of CNS neurons and is required for dendrite outgrowth. (A) Immunohistochemical analysis of retrolinkin (labeled as RTLN) expression in the brain cortex, cerebellum, and dorsal root ganglia (DRG) of adult mouse. Regions within white boxes (top) are shown at higher magnification (bottom). Note that retrolinkin was strongly expressed in the dendrites of hippocampus and the molecular layer enriched of Purkinje cell dendrites in cerebellum. Scale bar, 100 μm. (B) Cultured hippocampal neurons were fixed and immunostained for endogenous retrolinkin (green) and the dendritic marker MAP2 (red) or the axonal marker TAU1 (red). Scale bar, 10 μm. (C) Background-subtracted, mean intensity of retrolinkin fluorescence in primary axons and dendrites. Measurement of fluorescence intensity is expressed in arbitrary units per square area in both axons and dendrites. All images were obtained in the same settings below saturation at a resolution of 1024 × 1024 pixels (12 bits) (n = 16; ***p < 0.001). (D) Hippocampal neurons were transfected at DIV1 with shRNA constructs coexpressing red fluorescent protein (RFP) and shRNA or cotransfected with shRNA and RNAi-resistant expression constructs (indicated by asterisk) and fixed at DIV5, followed by immunostaining with the anti-RFP antibody. Scale bar, 100 μm. (E) Quantification of soma area and neurite length per transfected neuron. Shown are average soma area (± SEM) and neurite length (± SEM) of 40–60 neurons (*p < 0.05; **p < 0.01).

FIGURE 2:

Retrolinkin interacts with endophilin A1 (labeled as EEN1) directly. (A) Schematic representation of the domain structures of retrolinkin and endophilin A1. TM, transmembrane regions. (B) Yeast two-hybrid assay showing the interaction of retrolinkin (aa 31–574) with full-length (FL) endophilin A1. Interaction of the fusion proteins was detected by growth of yeast cells in the absence of adenine and histidine (−Ade, His) and the α-galactosidase colorimetric assay as described in Materials and Methods. (C) Lysates from adult mouse brain were subjected to coIP with antibodies to retrolinkin or endophilin A1, followed by Western blotting analysis. IB, immunoblotting. (D) In vitro binding between endophilin A1 and the N-terminus of retrolinkin. Recombinant His-tagged endophilin A1 was incubated with GST-fusion proteins conjugated to glutathione–Sepharose. GST alone and GST-βIII spectrin served as controls. (E) The retrolinkin-binding site maps to the SH3 domain of endophilin A1. Shown is a GST pull-down assay using His-tagged retrolinkin (aa 31–460) and GST-tagged endophilin A1 fragments. (F) The endophilin A1–binding site maps to the PRD domain of retrolinkin. Shown is a GST pull-down assay using lysates from 293 cells overexpressing myc-tagged endophilin A1. A GST tag was fused to each retrolinkin fragment at the N-terminus of the protein. (G) Cultured hippocampal neurons were immunostained with antibodies to retrolinkin and endophilin A1. Boxed regions are shown at higher magnification. Scale bar, 10 μm. (H, I) Representative examples of double immunogold labeling of retrolinkin (18 nm, solid triangle) and endophilin A1 (12 nm, open triangle). (H′, I′) Higher magnification of boxed areas in H and I, respectively. (J) Negative control with secondary antibodies only. Scale bar, 350 nm in H–J, and 100 nm in H′, I′. (K) Quantification of the colocalization between retrolinkin and endophilin A1 in immunoEM. Data represent means ± SEM (n = 3).

FIGURE 3:

Retrolinkin and endophilin A1 are required for BDNF-induced dendritic outgrowth and acute ERK activation. (A) Neurons transfected with shRNA constructs at DIV1 were cultured for 4 d in the presence of BDNF (25 ng/ml) and allowed to extend axons and dendrites. Scale bar, 100 μm. (B) Quantification of soma area and the length of dendrites per transfected neuron in A. Shown are average soma area (± SEM) and dendrite length (± SEM) of 40–60 neurons (*p < 0.05; ***p < 0.001). (C, D) Cortical neurons infected with Lentivirus expressing shRNA were treated with BDNF for different periods and lysed. Total and phosphorylated protein levels were examined by Western blotting. (E) Quantitative analysis of the immunoblot bands. Average pTrk, pERK1/2, pAkt, and pPLCγ levels were normalized to levels detected at the 0-min time point and represented as fold induction. Data represent means ± SEM (**p < 0.01, n = 5). (F) DIV10 hippocampal neurons were starved for 2 h, treated with 25 ng/ml BDNF, fixed, and immunostained with antibodies to pERK1/2 and retrolinkin or endophilin A1. (G) Quantification of the colocalization between pERK1/2 and endophilin A1 or retrolinkin. Data represent means ± SEM (n = 12–20) (*p < 0.05). (H) Same as F, except that neurons were immunostained with antibodies to pAkt and retrolinkin or endophilin A1. Scale bar, 10 μm. EEN1, endophilin A1; RTLN, retrolinkin.

FIGURE 4:

Retrolinkin is required for BDNF-triggered TrkB internalization. (A) DIV10 hippocampal neurons were starved for 2 h and incubated with 25 ng/ml BDNF for 30 min, fixed, and immunostained with antibodies to pTrk and retrolinkin or endophilin A1. Scale bar, 10 μm. (B, C) Representative examples of double immunogold labeling of adult mouse hippocampus with antibodies to pTrk (18 nm) and retrolinkin (12 nm). (B′, C′) Higher magnification of boxed areas in B and C, respectively. (D, E) Representative examples of double immunoEM labeling of pTrk (18 nm) and endophilin A1 (12 nm). (D′, E′) Higher magnification of boxed areas in D and E, respectively. (F) Negative control with secondary antibodies only. Scale bar, 350 nm in B–F, 100 nm in B′–E′. (G) Quantification of the colocalization between pTrk and retrolinkin in immunoEM. Data represent means ± SEM (n = 3). (H) Quantification of the colocalization between pTrk and endophilin A1 in immunoEM. Data represent means ± SEM (n = 3). (I) Cortical neurons infected with Lentivirus expressing shRNA were treated with BDNF (25 ng/ml) for 30 min. TrkB internalization was then measured by cleavable surface biotinylation. Surface TrkB was detected by Western blotting with anti-panTrk. Total TrkB and tubulin were detected by Western blotting of whole-cell lysates. The quantified immunoblot bands are shown (right). Values were normalized to the respective TrkB levels of control (**p < 0.01, n = 3). (J) Hippocampal neurons transfected with FLAG-TrkB and shRNA expression constructs were treated with BDNF (25 ng/ml) for 30 min. Internalized TrkB was labeled with FITC–α-FLAG and analyzed by confocal microscopy. (K) Hippocampal neurons transfected with shRNA constructs for 3–4 d were starved for 2 h, followed by incubation with 10 μg/ml Alexa Fluor 488–conjugated transferrin for 1 h. Neurons were fixed and analyzed by confocal microscopy. EEN1, endophilin A1; RTLN, retrolinkin.

FIGURE 5:

Activated TrkB traffic through APPL1 endosomes. (A) Localization of pTrk, pERK1/2, and pAkt to APPL1-positive endosomes. DIV10 hippocampal neurons were starved for 2 h, stimulated with BDNF (25 ng/ml) for 30 min, and double stained with different antibodies. Scale bar, 10 μm. (B, C) Representative examples of double immunogold labeling of adult mouse hippocampus with antibodies to pTrk (18 nm) and APPL1 (12 nm). (D) Negative control with secondary antibodies only. (B′–D′) Higher magnification of boxed areas in B–D, respectively. Scale bar, 350 nm in B–D, 100 nm in B′–D′. (E) Quantification of the colocalization of pTrk with APPL1 in immunoEM. Data represent means ± SEM (n = 3–5). (F) CoIP of lysates of HEK293 cells overexpressing FLAG-tagged TrkB and EGFP-APPL1 with immobilized FLAG antibody. Immunoblot was probed with antibodies to FLAG and GFP. (G, H) The colocalization of retrolinkin or endophilin A1 with APPL1 was increased upon BDNF treatment. DIV10 hippocampal neurons were starved for 2 h, stimulated with BDNF (25 ng/ml) for 30 min, and double stained with antibodies to APPL1 and retrolinkin or endophilin A1. Scale bar, 10 μm. (I) Quantification of the colocalization between APPL1 and retrolinkin or endophilin A1. Data represent means ± SEM (n = 12–20) (*p < 0.05; ***p < 0.001). (J, K) Representative examples of double immunogold labeling of adult mouse hippocampus with antibodies to retrolinkin (18 nm) and APPL1 (12 nm). (L, M) Representative examples of double immunogold labeling with antibodies to endophilin A1 (12 nm) and APPL1 (18 nm). (N) Negative control with secondary antibodies only. (J′–N′) Higher magnification of boxed area in J–N, respectively. Scale bar, 350 nm in J–N, 100 nm in J′–N′. (O) Quantification of the colocalization of retrolinkin or endophilin A1 with APPL1 in immunoEM. Data represent means ± SEM (n = 3–5). EEN1, endophilin A1; RTLN, retrolinkin.

FIGURE 6:

Retrolinkin and endophilin A1 are required for BDNF-induced acute ERK activation from early endosomes. (A) Hippocampal neurons transfected with shRNA constructs were treated with BDNF (25 ng/ml) for 30 min, fixed, and immunostained with antibodies to pTrk, APPL1, and RFP. (B) Quantification of the colocalization between pTrk and APPL1 in A. Data represent means ± SEM (**p < 0.005, n = 30–35). (C) Cortical neurons infected with Lentivirus expressing shRNA were stimulated with BDNF (25 ng/ml) and subjected to subcellular fractionation. The early (fractions 1 and 2) and late (fractions 3 and 4) endosomal fractions were extracted after centrifugation with an Opti-Prep discontinuous gradient and analyzed by immunoblotting. Rab5 and EEA1 served as markers for early endosomes, and Rab7 served as a marker for late endosomes. (D) Quantitative analysis of the immunoblot bands in C. Data represent means ± SEM (**p < 0.01; ***p < 0.001, n = 3). EEN1, endophilin A1; n.s., not significant; RTLN, retrolinkin.

FIGURE 7:

Recruitment of endophilin A1 to APPL1 endosomes requires retrolinkin activity. (A) Hippocampal neurons transfected with the retrolinkin shRNA construct were starved for 2 h, treated with BDNF (25 ng/ml), fixed, and immunostained with antibodies to pTrk, endophilin A1, and RFP. Quantification of colocalization is shown (right). Data represent means ± SEM (n = 15) (**p < 0.01). (B) Hippocampal neurons transfected with the endophilin A1 shRNA construct were stimulated with BDNF and immunostained with antibodies to pTrk, retrolinkin, and RFP. (C) Quantification of colocalization between pTrk and endophilin A1 or retrolinkin, respectively. Data represent means ± SEM (n = 15). Scale bar, 10 μm. (D) CoIP of HEK293 cells overexpressing EGFP-APPL1 and FLAG-TrkB or FLAG-endophilin A1. Cell lysates were incubated with immobilized FLAG antibody. Immunoblot was probed with antibodies to FLAG and GFP. (E) Hippocampal neurons transfected with control or retrolinkin shRNA construct were immunostained with antibodies to endophilin A1, APPL1, and RFP after BDNF treatment. (F) Quantification of the colocalization between endophilin A1 and APPL1. Data represent means ± SEM (***p < 0.001; n = 15). (G) Hippocampal neurons transfected with the control or endophilin A1 shRNA construct were immunostained with antibodies to retrolinkin, APPL1, and RFP after BDNF treatment. Scale bar, 10 μm. (H) Quantification of the colocalization between retrolinkin and APPL1. Data represent means ± SEM (n = 15). EEN1, endophilin A1; RTLN, retrolinkin.

FIGURE 8:

Working model for a role of retrolinkin and endophilin A1 in BDNF–TrkB endocytic trafficking and downstream signaling pathways. TrkB receptors are activated upon binding to BDNF, and the internalization of BDNF–TrkB complex into endocytic vesicles is mediated by retrolinkin through unknown mechanisms. After TrkB-positive endocytic vesicles mature into APPL1 endosomes, endophilin A1 is recruited to these endosomes by retrolinkin, and then downstream effectors such as ERK1/2 are recruited to signaling endosomes for acute activation. EEN1, endophilin A1; RTLN, retrolinkin.