Bicaudal-D1 regulates the intracellular sorting and signalling of neurotrophin receptors

Abstract

We have identified a new function for the dynein adaptor Bicaudal D homolog 1 (BICD1) by screening a siRNA library for genes affecting the dynamics of neurotrophin receptor-containing endosomes in motor neurons (MNs). Depleting BICD1 increased the intracellular accumulation of brain-derived neurotrophic factor (BDNF)-activated TrkB and p75 neurotrophin receptor (p75(NTR)) by disrupting the endosomal sorting, reducing lysosomal degradation and increasing the co-localisation of these neurotrophin receptors with retromer-associated sorting nexin 1. The resulting re-routing of active receptors increased their recycling to the plasma membrane and altered the repertoire of signalling-competent TrkB isoforms and p75(NTR) available for ligand binding on the neuronal surface. This resulted in attenuated, but more sustained, AKT activation in response to BDNF stimulation. These data, together with our observation that Bicd1 expression is restricted to the developing nervous system when neurotrophin receptor expression peaks, indicate that BICD1 regulates neurotrophin signalling by modulating the endosomal sorting of internalised ligand-activated receptors.

Keywords: Bicd1; TrkB; intracellular sorting; neurotrophin signalling; p75NTR.

Figure 1. Validation of Bicaudal D homolog 1 (Bicd1) gene-trapped embryonic stem (ES) cells

A Lateral (A′) and dorsal (A″) views of an X-gal-stained E12.5 embryo derived from RRP227 Bicd1^gt/+ ES cells, demonstrating Bicd1 expression throughout the developing nervous system, but particularly strong in the spinal cord (SC), hindbrain and dorsal root ganglia (DRG, asterisks). Scale bars, 1 mm. Removing the head at the cervical region and visualising the cut surface (A‴) shows that Bicd1 expression is particularly high in the ventral horns of the spinal cord (white arrowheads), DRG and ventral nerve tracts descending from these structures (black arrowheads). Scale bar, 200 μm. B Paraffin-embedded transverse section taken from the thoracic region of the embryo shown in (A), immunostained for HB9 and counterstained with Nile red. HB9 protein (brown) is localised exclusively in ventral horn motor neuron nuclei (MN), whilst Bicd1-lacZ (blue) is expressed both in MNs and adjacent DRG. Scale bar, 100 μm. C, D Transverse section of a spinal cord of a E12.5 wild-type mouse embryo immunostained for BICD1 (green) and Islet1 (red). Images taken from regions approximated by the black boxes in (B) show intense immunoreactivity for BICD1 in nerve tracts (C; central box in B) and in a subset of Islet1-positive DRG nuclei (D; right side box in B). Scale bars, 20 μm. E Western blotting of whole-cell lysates generated from Bicd1^gt/gt and wild-type control ES cell-derived MNs showing relative expression levels of BICD1, TrkB, p75^NTR and β-actin as a loading control. F Quantification of western blots for BICD1, pan-Trk (correspondent to TrkB and TrkC receptors in these samples) and p75^NTR normalised to βIII tubulin (n = 3, paired t-test, mean ± s.e.m., *P < 0.05, ***P < 0.001; n.s., not significant). Source data are available online for this figure.

Figure 2. Internalisation of the binding fragment of tetanus toxin (H_CT) in Bicd1^gt/gt motor neurons (MNs)

A, B AlexaFluor555-conjugated H_CT was internalised for 1 h at 37°C by wild-type and Bicd1^gt/gt MNs, which were then acid-washed and fixed. (A) Representative pseudo-coloured images of wild-type (top) and Bicd1^gt/gt (bottom) MNs were used to generate a heat map profile to better visualise the difference in relative amounts of internalised H_CT between the two genotypes. Scale bar, 20 μm. (B) Quantification of H_CT internalised by wild-type and Bicd1^gt/gt MNs after 1 h incubation at 37°C (n = 3, t-test, mean ± s.e.m., **P < 0.01). C Quantification of H_CT internalised by Bicd1^gt/gt MNs overexpressing GFP or BICD-GFP for 1 h at 37°C. Note that BICD1-GFP overexpression significantly decreased H_CT accumulation (red squares; 35–40 transfected cells were quantified per genotype; Mann–Whitney test, **P < 0.01). D Gold-conjugated H_CT (10 nm) was internalised for 1 h at 37°C by wild-type and Bicd1^gt/gt MNs, which were then fixed and processed for transmission electron microscopy. In Bicd1^gt/gt MNs, colloidal gold-H_CT (arrowheads) accumulated in different types of organelles, which were classified as MVBs, endosomes containing membranes (‘membranous’), endosomes with amorphous content (‘amorphous’) and tubular endosomes (‘tubular’). Scale bar, 200 nm. E Quantification of the relative abundance of each sub-type of colloidal gold-H_CT-containing organelle for each genotype (n = 2, mean ± s.e.m.).

Figure 3. Sorting nexin 1 (SNX1) co-localises with TrkB and the binding fragment of tetanus toxin (H_CT)

1. H_CT and TrkB co-localise in wild-type motor neurons (MNs). AlexaFluor555-conjugated H_CT (red) was internalised for 1 h at 37°C; neurons were then acid-washed, fixed and immunostained for TrkB (green). TrkB/H_CT-positive structures (arrowheads) were frequently detected in the cell soma and neurites. Scale bar, 5 μm.

2. N2A neuroblastoma cells over-expressing FLAG-TrkB were incubated with FLAG antibody and brain-derived neurotrophic factor (BDNF) for 1 h at 37°C and then acid-washed, fixed and immunostained to detect FLAG-TrkB (green) and endogenous SNX1 (red). Scale bar, 10 μm.

3. Quantification of FLAG-TrkB/SNX1 co-localisation (n = 3, 31 cells in total analysed).

4. N2A cells overexpressing FLAG-TrkB were incubated with FLAG antibody and BDNF-mCherry (red) for 1 h at 37°C and then acid-washed, fixed and immunostained to detect FLAG-TrkB (green) and endogenous Bicaudal D homolog 1 (BICD1) (blue). Note the presence of triple positive structures demonstrating that endogenous BICD1 associates with internalised TrkB-BDNF complexes. Scale bar, 10 μm.

Figure 4. Bicd1^gt/gt motor neurons accumulate H_CT, TrkB and p75^NTR in enlarged endosomal structures

1. Wild-type MNs were allowed to internalise gold-conjugated H_CT (10 nm, empty arrowheads), αTrkB (20 nm, white arrowheads) and αp75^NTR (5 nm, black arrowheads) in the presence of BDNF for 2 h at 37°C and then processed for transmission electron microscopy. Colloidal gold-conjugated probes were found in a variety of organelles ranging from tubular endosomes (Aa), to MVBs (Ab, Bc) to endosomes containing membranes (Ac). These organelles are pseudocoloured according to the number of probes that they contain: yellow, pink and green representing single, double and triple labelled compartments, respectively. Scale bars, 200 nm (main panels); 50 nm (insets).

2. Bicd1^gt/gt MNs were treated as in (A) and then processed for transmission electron microscopy. Notably, in addition to the previously described organelles, colloidal gold-conjugated probes were strongly associated with endosomes with “amorphous” content (Ba, Bb). In addition, double and triple labelled enlarged organelles were more prevalent in Bicd1^gt/gt motor neurons compared to wild-type cells. Scale bars, 200 nm (main panels); 50 nm (insets).

Figure 5. Bicd1^gt/gt motor neurons (MNs) show increased brain-derived neurotrophic factor (BDNF)-dependent intracellular accumulation of TrkB, which is unaffected by treatment with lysosomal protease inhibitor

1. TrkB antibody (αTrkB) was internalised for 1 h at 37°C by wild-type and Bicd1^gt/gt MNs, which were then acid-washed, fixed and immunostained for βIII tubulin and AlexaFluor-conjugated anti-rabbit IgG to detect αTrkB. Scale bar, 20 μm.

2. Quantification of internalised αTrkB from three independent experiments (n = 3, t-test, mean ± s.e.m.; n.s., not significant).

3. Internalisation of αTrkB as described for (A), but stimulated with 100 ng/ml of BDNF. Scale bar, 20 μm.

4. Quantification of internalised αTrkB in the presence of BDNF from three independent experiments (n = 3, t-test, mean ± s.e.m., **P < 0.01).

5. Wild-type and Bicd1^gt/gt MNs were co-incubated with αTrkB, 100 ng/ml of BDNF and a cocktail of lysosomal inhibitors (leupeptin 200 μM, E64D 2 μM, pepstatin A 20 μM) or DMSO vehicle control for 15 min at 37°C. Unbound antibody was then removed by repeated washing before chasing the internalised αTrkB pool under identical conditions. Cells were subsequently lysed at different time points and αTrkB captured on protein-G-conjugated magnetic beads followed by western blotting for TrkB. Note that the truncated TrkB.T1 isoform was significantly enriched relative to TrkB.FL in Bicd1^gt/gt MNs (lower panel) compared to wild-type controls (upper panel). Inhibition of lysosomal proteases prevented αTrkB.FL degradation in wild-type MNs (compare with DMSO-treated samples), but was ineffective in Bicd1^gt/gt cells.

6. Wild-type and Bicd1^gt/gt MNs were co-incubated with αTrkB, 100 ng/ml of BDNF and lysosomal inhibitors as described in (E) for 15 min at 37°C in the presence or absence of 10 μM MG132. Upon cell lysis, αTrkB was captured as above. Immunoprecipitated samples were probed for TrkB (upper panels), with the FK2 antibody, which targets mono- and poly-ubiquitinated proteins (middle panels), and with horseradish peroxidase-conjugated anti-rabbit immunoglobulins in order to detect internalised αTrkB. TrkB.T1 was enriched in Bicd1^gt/gt MNs in both conditions, but TrkB ubiquitination was detected only in Bicd1^gt/gt MN samples treated with MG132.

Source data are available online for this figure.

Figure 6. Bicd1^gt/gt motor neurons (MNs) have increased cell surface levels of TrkB.T1

1. Wild-type and Bicd1^gt/gt MNs were fixed and immunostained without permeabilisation to detect cell-surface-exposed TrkB using the TrkB antibody used in Fig5. Scale bar, 20 μm.

2. Quantification of cell-surface-localised TrkB receptors from three independent experiments (n = 3, t-test, mean ± s.e.m., *P < 0.05).

3. Representative western blotting of TrkB isoforms present on the surface of wild-type and Bicd1^gt/gt MNs at steady state. Cell surface proteins were biotinylated, purified on neutravidin sepharose beads and probed for the extracellular domain of TrkB. SOD1 was used as a control for cytosolic proteins. The input (INP), supernatant (SN) and biotinylated cell surface protein (beads) fractions are shown. Note the significantly increased level of TrkB.T1 relative to TrkB.FL in Bicd1^gt/gt MNs compared to wild-type controls.

4. Quantification of cell-surface-biotinylated TrkB.FL and TrkB.T1 receptors in (C) (t-test; mean ± s.e.m.; **P < 0.001, n.s., not significant, n = 4).

5. TrkB.FL/TrkB.T1 receptor ratio (t-test; mean ± s.e.m.; **P < 0.001, n = 4).

Source data are available online for this figure.

Figure 7. AKT and ERK1/2 phosphorylation are altered in brain-derived neurotrophic factor (BDNF)-stimulated Bicd1^gt/gt motor neurons (MNs)

A Starved wild-type and Bicd1^gt/gt MNs were stimulated with 100 ng/ml BDNF for various time points before cell lysis and immunoblotted for phospho-AKT (pAKT; S473) and then re-probed for total AKT. B Densitometric analysis of pAKT (S473) from three independent experiments including the one shown in (A). pAKT band densities were normalised to total AKT (pAKT/AKT) for each time point and plotted as the mean ± s.e.m. Reduced phosphorylation of AKT in Bicd1^gt/gt MNs compared to wild-type controls was statistically significant at all time points (two-way ANOVA,P < 0.0001). C Starved wild-type and Bicd1^gt/gt MNs were stimulated with 100 ng/ml BDNF for various time points before lysis and immunoblotted for phospho-ERK1/2 (pERK1/2) and then re-probed for total ERK1/2. D Densitometric analysis of pERK1/2 from three independent experiments including the one shown in (C). pERK1/2 band densities were normalised to total extracellular signal-regulated kinase (ERK; pERK/ERK) for each time point and plotted as the mean ± s.e.m. Reduced phosphorylation of ERK1/2 in Bicd1^gt/gt MNs compared to wild-type controls was statistically significant at all time points (two-way ANOVA,P < 0.0001). E, F Starved wild-type and Bicd1^gt/gt MNs were stimulated with 100 ng/ml BDNF for 10 min before cell lysis. Samples were immunoblotted for phospho-TrkB (pTrkB; top panel), phospho-AKT (pAKT; S473; middle panels) and pERK1/2 (lower panels) (E). The same lysates were probed for total TrkB, AKT and ERK1/2 to assess loading parity between samples (F). G Starved wild-type (left panels) and Bicd1^gt/gt MNs (right panels) were stimulated with 100 ng/ml BDNF for different times before cell lysis. Samples were immunoblotted for phospho-AKT (pAKT; S473) and total AKT. Note that there was a gradual decrease in pAKT signal over time for wild-type cells, which contrasted with sustained pAKT levels for Bicd1^gt/gt MNs. Source data are available online for this figure.

Figure 8. Proposed role of BICD1 in neurotrophin receptor trafficking and signalling

In wild-type motor neurons (MNs), brain-derived neurotrophic factor (BDNF) binds to and activates TrkB. Ligand–receptor complexes are internalised at synaptic sites located in the periphery (1); note that for clarity, internalisation of these complexes from the plasma membrane of the cell body is not depicted. Ligand–receptor complexes are sorted to signalling endosomes (2), retrogradely transported in a cytoplasmic dynein-dependent process (3), towards the cell soma where they associate with somatic sorting endosomes (4) decorated by sorting nexin 1 (SNX1) and other retromer components. Different neurotrophin receptor pools are then trafficked towards MVB/lysosomes (5a) or the proteasome (5b) for degradation, or recycled back to the plasma membrane (5c). Impairment of the lysosomal targeting of TrkB in cells lacking BICD1 is envisaged to impair the flow of the receptor from somatic sorting endosomes towards lysosomes and redirect them either to the recycling route back to the plasma membrane or to the proteasome for ubiquitin-mediated degradation. The main consequence of these mis-sorting steps is the increased accumulation of neurotrophin receptors on the cell surface at steady state. Such a chronic imbalance in receptor recycling over receptor degradation in Bicd1^gt/gt MNs is predicted to result in prolonged receptor activation after internalisation and/or overstimulation from repeated recycling to the plasma membrane. The increased levels of cell surface TrkB.T1 in Bicd1^gt/gt MNs (6) may be an adaptive response to overstimulation and serves to reduce BDNF-mediated activation of TrkB.FL and associated AKT (6) and ERK1/2 (7) signalling pathways.