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 (p75NTR) 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 p75NTR 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.
The dynein adaptor Bicaudal D homolog 1 (BICD1) modulates endosomal trafficking and signalling of ligand‐activated neurotrophin receptors in motor neurons. Depletion of BICD1 disrupts the endosomal sorting of BDNF‐activated TrkB by reducing its lysosomal degradation in favour of proteasomal targeting and promotes the re‐cycling of non‐degraded receptor to the plasma membrane.
BICD1 and neurotrophin receptors are co‐expressed in the developing nervous system.
BICD1 modulates the endocytic sorting of specific membrane cargoes in neurons.
Ligand‐dependent somatic sorting of neurotrophin receptor complexes requires BICD1.
BICD1 depletion alters BDNF signalling responses.
Neurons have complex axonal and dendritic arborisations, which are paramount for the function of the nervous system. The development and maturation of these extended neuronal networks require tight regulation of the intracellular transport of organelles and cargoes, such as mRNA, mitochondria, growth factor receptors and their signalling adaptor molecules, which in turn are necessary to ensure neuronal growth, differentiation and survival (Salinas et al, 2008; Hirokawa et al, 2010; Ascano et al, 2012).
Neurotrophins are essential for the development and maintenance of the nervous system (Ascano et al, 2012). The neurotrophin family consists of four structurally related growth factors: nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), NT3 and NT4/5 (Bibel & Barde, 2000; Huang & Reichardt, 2001). These ligands bind and activate the tropomyosin‐related kinases TrkA, TrkB and TrkC either alone or in combination with p75 neurotrophin receptor (p75NTR), which lacks enzymatic activity (Simi & Ibanez, 2010). Neurotrophin receptors have different binding specificities for their ligands: TrkA binds preferentially to NGF; TrkB to BDNF and NT4/5; and TrkC to NT‐3, whilst p75NTR can bind all four neurotrophins, and their precursors, the pro‐neurotrophins (Teng et al, 2010). The considerable crosstalk in the binding of neurotrophins to their receptors generates a variety of signalling outputs resulting in diverse cellular responses, which are initiated both by ligand availability and the relative abundance of each neurotrophin receptor at specific locations on the neuronal surface (Reichardt, 2006; Ascano et al, 2012). This signalling diversity is further enhanced because the same neurotrophin can have opposing effects, depending on whether it is presented to the neuron as the precursor or mature form (Teng et al, 2010). It is therefore important that neurotrophin signalling is tightly controlled both spatially and temporally, particularly in the developing nervous system when neurotrophins and their receptors are abundantly expressed.
The signalling cascades activated by neurotrophins in the neuronal periphery are regulated by the endocytosis, sorting and trafficking of neurotrophin receptors along the axon or dendrites towards the cell body, where they elicit transcriptional responses (Ibanez, 2007; Ascano et al, 2012; Schmieg et al, 2014). We have previously shown that the binding fragment of tetanus neurotoxin (HCT) is a reliable probe to monitor neurotrophin receptor uptake and intracellular trafficking (Bercsenyi et al, 2013). Indeed, HCT and p75NTR bound to NGF accumulate in clathrin‐coated pits and are internalised by a clathrin‐mediated endocytic pathway in motor neurons (MNs) (Deinhardt et al, 2006a, 2007). Furthermore, HCT shares the same axonal retrograde transport organelles with neurotrophins and their receptors (Lalli & Schiavo, 2002; Deinhardt et al, 2006b).
Since a comprehensive understanding of the molecular mechanisms controlling neurotrophin receptor internalisation and trafficking is still lacking, we sought to identify new genes involved in this pathway by performing an small interfering RNA (siRNA) screen in MNs derived from transgenic HB9‐GFP embryonic stem (ES) cells using HCT and an antibody directed against the extracellular domain of p75NTR (αp75NTR) as fluorescent reporters (Terenzio et al, 2014). Importantly, expression of green fluorescent protein (GFP) driven by the Hb9 homeobox gene enhancer (Wichterle et al, 2002) facilitated the unequivocal identification of MNs generated from these ES cells upon differentiation (Terenzio et al, 2014).
Using this approach, we identified a small cohort of genes affecting the intracellular accumulation of HCT and αp75NTR when silenced (see Supplementary Table S1). Knockdown of one gene in particular, Bicaudal D homolog 1 (Bicd1), revealed an increased internalisation phenotype for HCT and was selected for further analyses, which demonstrated that BICD1 depletion also increased the intracellular accumulation of ligand‐bound p75NTR and TrkB (Terenzio et al, 2014).
Bicaudal D homolog 1 is known to participate in endosomal trafficking and dynein‐mediated processes (Hoogenraad et al, 2001; Matanis et al, 2002; Bianco et al, 2010; Aguirre‐Chen et al, 2011), including retrograde transport in neuronal cells (Wanschers et al, 2007). Furthermore, BICD1 has important roles in the development and function of the Drosophila and Caenorhabditis elegans nervous systems (Li et al, 2010). We now show that BICD1 is a key regulator of the intracellular trafficking of neurotrophin receptors and that it performs this role by controlling the BDNF‐triggered sorting and progression of p75NTR and TrkB through the endosomal pathway. Thus, BICD1 depletion disrupted this mechanism to favour p75NTR and TrkB recycling over lysosomal degradation, which in turn affected signalling responses to BDNF stimulation.
Our data identify a novel function for BICD1 as a modulator of neurotrophin receptor dynamics and signalling, which may also be relevant for other receptor tyrosine kinases in different cellular systems.
Bicd1 is strongly expressed in the developing central and peripheral nervous systems
One of the most prominent candidates from our siRNA screen was BICD1 (Terenzio et al, 2014). Since BICD1 and related proteins are cytoplasmic dynein adaptors (Hoogenraad et al, 2001; Matanis et al, 2002), and BICD1 has been postulated to function in retrograde transport (Wanschers et al, 2007), we decided to focus our efforts on addressing the role played by BICD1 in the regulation of neurotrophin receptor trafficking in our model system.
We used a Bicd1 gene‐trapped ES cell clone (Bicd1gt/+; gene trap RRP227) for the functional characterisation of BICD1. This gene trap vector expressed a β‐galactosidase cassette, allowing us to determine Bicd1 expression patterns in chimeric embryos generated from Bicd1gt/+ ES cells (Fig 1A). X‐gal histochemistry of Bicd1gt/+ ES cell‐derived embryos revealed that at embryonic day 12 (E12), Bicd1 was highly and almost exclusively expressed in ventral horn MNs of the developing spinal cord (Fig 1A′–A‴), dorsal root ganglia (DRG; Fig 1A″–A‴) and brain (Fig 1A′ and A″). These X‐gal‐stained embryos were then paraffin embedded, cross‐sectioned and immunostained to reveal that Bicd1 expression was highest in HB9‐positive ventral horn MNs (Fig 1B), a sub‐population of DRG neurons (Fig 1D) and in the nerve tracts emanating from these structures (Fig 1A‴,C). High Bicd1 expression in the developing nervous tissue closely matched the pattern of immunoreactivity for BDNF, Trk receptors and p75NTR (Supplementary Fig S1A–C). Altogether, these observations suggest that BICD1 plays a role in the developing nervous system at a time when neurotrophins and their receptors are highly expressed (Davies, 1994; Klein, 1994; Ernfors, 2001).
However, only one developmental day later, the expression pattern of Bicd1 had dramatically changed: at E14.5, Bicd1‐LacZ had been lost from the brain (Supplementary Fig S1E) and spinal cord (Supplementary Fig S1F), yet was still strongly retained in DRG (Supplementary Fig S1F and G) and was most notably upregulated in skin, skeletal muscle and heart ventricles, but interestingly, not in the atria (Supplementary Fig S1H).
Bicd1gt/gt ES‐cell‐derived MNs are a reliable in vitro model system to study BICD1 function
We had planned to use Bicd1gt/+ × C57/Bl6 mouse chimeras to generate a Bicd1gt/+ founder colony. However, mating of male chimeras with approximately 75% ES cell contribution to wild‐type females produced no mutant offspring, indicating that germline transmission of the Bicd1gt/+ allele had failed to take place. We therefore selected for loss of heterozygosity in culture using high G418 concentrations (Lefebvre et al, 2001). Resultant homozygous Bicd1gt/gt ES cells were isolated, expanded and differentiated into MNs.
BICD1 transcript and protein levels were reduced by approximately 70% in Bicd1gt/gt MNs compared to wild‐type cells (Fig 1E and F; Supplementary Fig S1D). Complete ablation of Bicd1 expression was not expected since gene trap insertions are prone to unpredicted downstream mRNA splicing events (Voss et al, 1998). In contrast, Bicd2 mRNA levels were found to be approximately 80% higher in Bicd1gt/gt MNs compared to wild‐type controls (Supplementary Fig S1D), suggesting that BICD2 might compensate for the partial loss of BICD1. However, this scenario is unlikely since BICD2 protein levels were not increased in Bicd1gt/gt MNs (Supplementary Fig S2A).
BICD1 depletion had no significant effect on the expression of the MN markers Hb9 and ChAT (choline acetyltransferase; Supplementary Fig S1D), or p75NTR (Fig 1E and F; Supplementary Fig S1D), whilst Trk protein levels showed an approximate 30% decrease using a pan‐Trk antibody (Fig 1F). Since MNs do not express TrkA, this reduction could only be attributed to decreased levels of TrkB and/or TrkC. Accordingly, a similar decrease of TrkB transcript levels was observed (Supplementary Fig S1D).
Importantly, Bicd1gt/gt MNs were indistinguishable from their wild‐type counterparts, since we observed no statistically significant difference in terms of either the abundance of synaptic boutons (Supplementary Fig S2B and C) or the morphology of the neurite network (Supplementary Fig S2D–G). Taken together, these results demonstrated that RRP227 gene trap ES‐cell‐derived MNs were a suitable model system with which to investigate the functional consequences of BICD1 depletion on the trafficking of neurotrophin receptors.
BICD1 depletion affects the internalisation and intracellular fate of HCT
Our siRNA screen revealed that Bicd1 knockdown increased the intracellular accumulation of HCT compared to cells transfected with control siRNA (Terenzio et al, 2014), a phenotype that was replicated in Bicd1gt/gt MNs (Fig 2A and B). Overexpressing BICD1‐GFP in Bicd1gt/gt MNs rescued this phenotype by decreasing HCT accumulation compared to neurons expressing GFP alone (Fig 2C and Supplementary Fig S3A), confirming that the observed phenotype was a direct consequence of BICD1 depletion. To further investigate the nature of the intracellular compartment to which BICD1 associates, we purified a population of endosomes containing monocrystalline ion oxide nanoparticles (MION)‐conjugated HCT (Deinhardt et al, 2006b; Wade et al, 2012). Many of these organelles are signalling endosomes as they are known to contain Trk receptors and p75NTR and are transported from the periphery towards the MN soma by cytoplasmic dynein (Schmieg et al, 2014). BICD1 associated with this compartment together with its known interactors p150Glued and p50, two subunits of the dynactin complex that are required for dynein motor activity, and Rab6, a small GTPase involved in Golgi to ER retrograde trafficking (Supplementary Fig S3B; Matanis et al, 2002; Fuchs et al, 2005).
Because BICD1 undergoes axonal retrograde transport (Terenzio et al, 2014) and has been previously implicated in regulating this dynein mediated process (Wanschers et al, 2007), we tested whether this trafficking pathway was altered in Bicd1gt/gt MNs. Surprisingly, there was no significant difference in the speed or frequency of axonal retrograde carriers containing αp75NTR (Supplementary Fig S3C) or HCT (Terenzio et al, 2014) between wild‐type and mutant neurons, suggesting that the increase in somatic accumulation of HCT observed in MNs depleted of BICD1 was not due to abnormalities in axonal retrograde transport.
Alternatively, the increased accumulation of HCT in Bicd1gt/gt MNs (Fig 2A and B) might have been caused by defective endosomal sorting and/or trafficking of this probe in the cell soma. To test this possibility, we used transmission electron microscopy to trace the intracellular fate of internalised colloidal gold‐conjugated HCT in wild‐type and Bicd1gt/gt MNs and determined the morphology of gold‐containing organelles at the ultrastructural level (Figs 2D and 4). Some of these HCT‐containing compartments resembled multi‐vesicular bodies (MVBs), which was not surprising since HCT was previously reported to associate with these organelles (Parton et al, 1987). Other HCT‐containing membrane compartments were described on the basis of their contents: ‘amorphous’ (no clearly identifiable content and low electron density), ‘membranous’ (relatively high membrane content) and ‘tubular’, because of their characteristic morphology (Fig 2D). Compared to wild‐type cells, Bicd1gt/gt MNs contained significantly more HCT in the ‘amorphous’ compartments where it clustered close to the limiting membrane (Fig 2D). Interestingly, when compared to wild‐type controls, Bicd1gt/gt MNs displayed an inverse relationship in the abundance of HCT‐positive ‘amorphous’ organelles relative to MVBs (Fig 2E). Occasionally, HCT concentrated as clusters within bud‐like protrusions, which were often contiguous with the lumen of compartments with ‘amorphous content’ (Supplementary Fig S3D). These structures were more commonly observed in Bicd1gt/gt MNs and we believe that they represent enlarged endosomal sorting compartments.
TrkB associates with enlarged endosomal compartments in Bicd1gt/gt MNs
Because HCT displayed an extensive co‐localisation with TrkB (Fig 3A) and p75NTR (Lalli & Schiavo, 2002; Deinhardt et al, 2006b, 2007) in wild‐type MNs, we inferred that neurotrophin receptors may also associate with enlarged endosomes in BICD1‐depleted cells. Electron microscopy of wild‐type and Bicd1gt/gt MNs incubated for 2 h with a cocktail of colloidal gold‐labelled probes: HCT (10 nm) together with antibodies directed against TrkB (20 nm) and p75NTR (5 nm) demonstrated that neurotrophin receptors and HCT were all present within a sub‐population of endosomes (Fig 4). As expected, the accumulation of all three probes within these organelles was greater in Bicd1gt/gt MNs compared to wild‐type cells (compare Fig 4A with 4B), indicating that endosomal trafficking of neurotrophin receptors was also affected by BICD1 depletion.
The increased association of HCT, TrkB and p75NTR with these compartments in Bicd1gt/gt MNs suggested that BICD1 could play a role in the endocytic sorting of neurotrophin receptors and HCT in neuronal cell bodies. This view was supported by observations that HCT co‐localised with components of the retromer complex, most notably sorting nexin 1 (SNX1; Supplementary Fig S4A and B) and Vps26 (Terenzio et al, 2014). TrkB was also localised to endogenous SNX1‐positive compartments (Fig 3B and C). We used N2A neuroblastoma cells overexpressing FLAG‐tagged TrkB for this experiment because of the inability to simultaneously detect endogenous TrkB and SNX1 using antibodies raised in the same species (see Supplementary Information). Furthermore, FLAG‐TrkB overexpression in N2A cells followed by incubation with BDNF‐mCherry demonstrated that endogenous BICD1 co‐localised with BDNF/TrkB‐positive organelles (Fig 3D).
Importantly, the co‐distribution of HCT with SNX1‐positive compartments was higher in Bicd1gt/gt MNs compared to wild‐type cells (Supplementary Fig S4A and B), suggesting that SNX1‐dependent sorting of HCT and neurotrophin receptors was affected by BICD1 depletion.
BICD1 depletion enhances the neurotrophin‐dependent intracellular accumulation of p75NTR and TrkB
We previously showed that p75NTR undergoes rapid recycling at the plasma membrane in the absence of neurotrophins (Deinhardt et al, 2007). This steady‐state recycling of p75NTR in the absence of exogenous ligands was unaffected by BICD1 depletion (Supplementary Fig S5A and B). To test whether BICD1 was required for the BDNF‐induced trafficking of p75NTR, we treated wild‐type and Bicd1gt/gt MNs with exogenous BDNF, a treatment which promotes the recruitment of p75NTR to a clathrin‐dependent endocytic pathway linked to axonal retrograde transport (Deinhardt et al, 2007). The addition of BDNF resulted in a marked increase in the intracellular accumulation of αp75NTR, which was significantly higher in Bicd1gt/gt MNs compared to wild‐type controls (Supplementary Fig S5C and D), indicating that BICD1 is required for the trafficking of BDNF‐bound p75NTR, but does not influence the neurotrophin‐independent trafficking of this receptor. This effect was unlikely to have resulted from any alteration in the axonal retrograde transport of αp75NTR, as this process was not affected by BICD1 depletion (Supplementary Fig S3C).
A similar ligand‐dependent accumulation phenotype was also observed for TrkB in Bicd1gt/gt MNs (Fig 5A and D). Like many receptor tyrosine kinases, activated TrkB‐BDNF complexes traffic to the late endosomal pathway and are ultimately delivered to lysosomes for degradation (Chen et al, 2005). We tested whether the increased accumulation of ligand‐activated TrkB in Bicd1gt/gt MNs was caused by the defective sorting of antibody‐bound TrkB to lysosomes, by inhibiting lysosomal proteases using a cocktail of well‐characterised inhibitors (leupeptin, E64D and pepstatin A). Under these conditions, TrkB accumulation was increased in wild‐type MNs, whereas the extent of TrkB accumulation in Bicd1gt/gt MNs was unaffected by this treatment (Supplementary Fig S6A). A similar trend was also established for αp75NTR accumulation (Supplementary Fig S6B). We did not detect any obvious difference either in lysosomal acidification or distribution of LAMP2‐positive organelles between Bicd1gt/gt and wild‐type MNs, suggesting that BICD1 depletion does not affect lysosome biogenesis or function, but is likely to cause an impaired sorting of neurotrophin receptors along the degradative pathway. Consistent with this idea was our observation that intracellular accumulation of HCT was unaffected by lysosomal inhibitors (Supplementary Fig S6C), which was not surprising since this probe does not enter the lumen of lysosomes (Supplementary Fig S6D) and therefore is not degraded by these organelles.
Since the results described in Supplementary Fig S6A and B were obtained by an imaging approach using fluorescent αTrkB and αp75NTR, we wanted to confirm these results using a more direct biochemical strategy. For this purpose, we selected a pulse‐chase protocol, which allowed us to follow the dynamics of a cohort of internalised receptors as they trafficked along the endosomal pathway. To this end, we loaded ES‐derived MNs with a 15‐min pulse of αTrkB in the presence of BDNF and lysosomal inhibitors or vehicle control, followed by 1 or 2 h incubation in the presence or absence of lysosomal inhibitors. After cell lysis, antibody pulldown followed by western blotting revealed a major band of full‐length TrkB (TrkB.FL; 145 kDa) and a minor band of TrkB.T1 (90 kDa), representing the truncated dominant‐negative form of this receptor (Fenner, 2012), in wild‐type cells (upper panel, Fig 5E). However, samples isolated from Bicd1gt/gt MNs displayed a striking increase of TrkB.T1 relative to TrkB.FL (lower panel, Fig 5E). In agreement with the immunofluorescence data quantified in Supplementary Fig S6A, lysosomal inhibitors proved effective in preventing the degradation of αTrkB in wild‐type, but not in Bicd1gt/gt MNs (Fig 5E), suggesting that BICD1 depletion impaired lysosome‐mediated degradation of these receptors.
TrkA is degraded by the combined actions of lysosomes and the proteasome (Sommerfeld et al, 2000; Geetha & Wooten, 2008), a pathway shared at least in part by TrkB (Sommerfeld et al, 2000). Interestingly, proteasomal inhibition redirects TrkA from late endosomes to the recycling route (Moises et al, 2009). Therefore, the residual degradation of TrkB by wild‐type MNs in the presence of lysosomal protease inhibitors at the latest time point (Fig 5E, 120 min) could have resulted from receptor clearance by the proteasome and suggested a possible explanation for the apparent ineffectiveness of lysosomal inhibitors in preventing the clearance of TrkB from Bicd1gt/gt MNs (lower panel, Fig 5E). To test this hypothesis and assess the impact of BICD1 on these degradative pathways, we repeated the 15‐min pulse step in the presence or absence of MG132, a specific proteasome inhibitor. Thus, probing the αTrkB immunoprecipitates with a TrkB antibody revealed profiles similar to those shown in the previous experiment (compare upper panels in Fig 5F with 5E). However, re‐probing the same membrane with a ubiquitin‐specific antibody revealed a signal only for the Bicd1gt/gt sample treated with a combination of lysosomal inhibitors and MG132 (Fig 5F). This result implied that within 15 min of exposure to BDNF, TrkB internalised by Bicd1gt/gt MNs was preferentially ubiquitinated and likely targeted for proteasome‐mediated degradation.
BICD1 depletion increases neurotrophin receptor levels at the plasma membrane
Inhibition of the proteasome, which causes an accumulation of ubiquitinated Trks, redirects TrkA from late endosomes to a plasma membrane recycling pathway (Moises et al, 2009). Depletion of BICD1 on the other hand led to the intracellular increase of HCT, TrkB and p75NTR in large vacuoles often connected to structures resembling tubular endosomes (Supplementary Fig S3D). Further insights into the nature of these organelles were provided by the co‐localisation of SNX1 with TrkB and HCT (Fig 3B and Supplementary Fig S4A and B), and the increase in SNX1‐associated HCT‐positive structures in Bicd1gt/gt MNs (Supplementary Fig S4A and B). Based on these findings and the notion that TrkB was preferentially ubiquitinated when BICD1 was depleted (Fig 5F), we decided to test whether the altered neurotrophin receptor trafficking observed in Bicd1gt/gt MNs resulted in an increased recycling of these receptors back to the plasma membrane.
In spite of TrkB‐FL transcript (Supplementary Fig S1D) and Trk protein levels (Fig 1E and F) being reduced in Bicd1gt/gt MNs relative to wild‐type cells, cell surface immunofluorescence and biotinylation experiments both demonstrated that significantly more TrkB was present on the plasma membrane of Bicd1gt/gt neurons compared to wild‐type controls (Fig 6A–E), with a similar trend also observed for p75NTR (Supplementary Fig S7A–D).
TrkB signalling is impaired in Bicd1gt/gt MNs
The most striking finding from the previous set of experiments was the substantial increase in TrkB.T1 localisation to the surface of Bicd1gt/gt MNs (Fig 6C–E). TrkB.T1 heterodimerises with TrkB.FL to inhibit auto‐phosphorylation and downstream signalling of the full‐length receptor (Eide et al, 1996; Fenner, 2012). Therefore, the substantially decreased ratio of TrkB.FL to TrkB.T1 (Fig 6E) on the plasma membrane of Bicd1gt/gt MNs, together with an increased cell surface localisation of p75NTR (Supplementary Fig S7A–D), would be expected to reduce phosphoinositide‐3 kinase (PI3K) and Ras‐mediated signal activation following treatment with BDNF.
To test this hypothesis, we stimulated MNs with BDNF for various time points before assaying lysates by western blotting for phosphorylated TrkB, AKT and ERK1/2. In wild‐type cultures, both AKT and ERK1/2 phosphorylation peaked at 10 min followed by a progressive attenuation of this response over the following 40 min. The same overall trend was observed for Bicd1gt/gt MNs, but phosphorylation of both AKT and ERK1/2 was lower at all time points tested (Fig 7A–D). Thus, even though more cell surface TrkB and p75NTR were available for ligand binding in Bicd1gt/gt MNs, this was accompanied by decreased activation of AKT and ERK1/2 following BDNF treatment. This reduced signalling response might be a consequence of decreased TrkB activation caused by the lower TrkB.FL to TrkB.T1 ratio present in Bicd1gt/gt MNs. This hypothesis was supported by data shown in Fig 7E–F, where phosphorylation of TrkB upon stimulation with BDNF for 10 min was lower in mutant MNs compared to wild‐type controls.
Proteosomal inhibition is known to induce sustained ERK1/2 activation following NGF stimulation (Moises et al, 2009), which was likely to result from enhanced TrkA recycling back to the plasma membrane. We now show that Bicd1gt/gt MNs respond to BDNF/TrkB activation through sustained PI3K/AKT signalling. Thus, whereas the phospho‐AKT (pAKT) profile tailed off at later time points in wild‐type cells, BICD1‐depleted MNs displayed a more prolonged AKT activation during 1 h of BDNF stimulation (Fig 7A–B). This trend was substantiated by prolonged stimulation experiments when phospho‐AKT levels, albeit lower, remained relatively stable over the entire time frame of the experiment in Bicd1gt/gt MNs (Fig 7G).
Using a novel siRNA‐screening approach designed to identify new players involved in the trafficking of neurotrophin receptors and neurotropic virulence factors in MNs (Terenzio et al, 2014), we identified several genes that affected the intracellular accumulation of HCT and αp75NTR. Because of our long‐standing interest in axonal retrograde transport, the most intriguing candidate revealed by this screen was Bicd1. This cytoplasmic dynein adaptor and closely related proteins, such as BICD2 and BICDR1, play diverse roles in nervous system development and maintenance (Matanis et al, 2002; Schlager et al, 2010). For instance, BICD2 is required for neuronal migration (Jaarsma et al, 2014), whereas BICD1 cooperates with dynein and kinesins in C. elegans nervous system patterning (Aguirre‐Chen et al, 2011). Furthermore, Drosophila BicD recycles clathrin heavy chain back to the plasma membrane during synaptic stimulation (Li et al, 2010) and controls distribution of Fragile X mental retardation protein (Bianco et al, 2010). Our in vivo observations now expand this list of functions by showing that BICD1 very likely plays an important role in the developing mouse nervous system, at least during the period when BDNF and neurotrophin receptors are highly expressed (Fig 1 and Supplementary Fig S1).
Silencing of Bicd1 increased the intracellular accumulation of HCT, and this was confirmed in MNs expressing the RRP227 Bicd1gt/gt hypomorphic allele. The intracellular distribution of HCT in Bicd1gt/gt MNs differed markedly from that observed in wild‐type cells (Fig 2D and E). In mutant cells, HCT clustered near the limiting membrane of a population of enlarged organelles with amorphous content, which lacked intra‐luminal vesicles typical of MVBs (Fig 2D) and was found in protrusions emanating from such structures (Supplementary Fig S3D). These observations suggested that endosomal sorting and/or maturation of these HCT‐containing compartments were perturbed in Bicd1gt/gt MNs. Because HCT is co‐transported with p75NTR and TrkB, we inferred that these receptors were also likely to be present in at least a sub‐population of the enlarged HCT‐labelled compartments present in Bicd1gt/gt MNs. This notion was supported by the findings that colloidal gold‐labelled HCT, αTrkB and αp75NTR all accumulated in the same enlarged organelles (Fig 4), which strongly suggested that these structures represented sorting compartments common to all three probes. Furthermore, the increased occurrence of such organelles in Bicd1gt/gt MNs indicated that BICD1 depletion affected the endosomal sorting of HCT, TrkB and p75NTR.
This hypothesis was supported by the observation that HCT‐positive organelles in Bicd1gt/gt MNs also displayed an increased co‐localisation with SNX1, a retromer component (Supplementary Fig S4A and B; Terenzio et al, 2014). Retromer is a large protein complex, which controls the endosome membrane re‐sculpturing process essential for the formation of transport carriers (Bonifacino & Hurley, 2008; Cullen & Korswagen, 2012) and the selection of specific cargoes (Harrison et al, 2014). Classically, this complex has been shown to mediate retrograde transport of membrane‐bound cargoes from endosomes to the trans‐Golgi network (TGN). However, Steinberg et al have recently shown that the retromer and specific SNX isoforms, such as SNX17 and SNX27, were involved in preventing lysosomal degradation in favour of maintaining plasma membrane levels of several transporters, signalling receptors and adaptor molecules, such as the glucose transporter GLUT1, PDGFRβ and the neurotrophin receptor‐binding protein Kidins220/ARMS (Steinberg et al, 2012, 2013). Moreover, retromer‐independent functions for SNX1 in the recycling of specific receptors to the plasma membrane have also been described (Nisar et al, 2010). The retrograde pathway to the TGN is unlikely to be involved in the re‐routing of HCT and neurotrophin receptors when BICD1 is depleted, since cholera toxin B subunit (CTB) trafficking to the Golgi was unaffected in Bicd1gt/gt MNs (Supplementary Fig S8). Importantly, this result also demonstrated that the depletion of BICD1 did not cause a general defect in endosomal trafficking.
Similar increased accumulation phenotypes in Bicd1gt/gt MNs were also observed for p75NTR and TrkB, but only after treatment with BDNF (Fig 5A–D and Supplementary Fig S5A–D). Upon stimulation, phosphorylated TrkB is mainly sorted to lysosomes and degraded (Chen et al, 2005; Bronfman et al, 2007; Huang et al, 2009). Our observations that the intracellular accumulation of p75NTR and TrkB was enhanced in Bicd1gt/gt MNs and that HCT appeared to be trapped in organelles resembling those shown to accumulate NGF in sympathetic neurons treated with lysosomal inhibitors (Claude et al, 1982) suggested that the trafficking of receptors normally targeted to lysosomes might be impaired by BICD1 depletion. Accordingly, inhibiting lysosomal proteases in wild‐type MNs phenocopied the increased accumulation of TrkB and p75NTR observed in Bicd1gt/gt cultures treated with BDNF (Supplementary Fig S6A and B). These data, together with the lack of overt alterations in lysosome morphology or activity in Bicd1gt/gt MNs, implied that BICD1 is not likely to be involved in lysosome biogenesis or function, but participates in a sorting step regulating the targeting of endocytic cargoes to this organelle (Fig 8). TrkB antibody feeding assays confirmed this view by demonstrating that upon treatment with BDNF, TrkB internalised by Bicd1gt/gt MNs was not protected from degradation in the presence of lysosome protease inhibitors (Fig 5E). This result suggested that TrkB‐BDNF complexes were preferentially degraded by an alternative mechanism in these cells. This was likely to be via the proteasome since ubiquitinated TrkB was detected only in immunoprecipitates retrieved from Bicd1gt/gt MNs in which proteasomal activity was inhibited (Fig 5F). Trk receptors have been shown to be ubiquitinated, and a proportion of this modified receptor pool is degraded by the proteasome (Sommerfeld et al, 2000; Geetha & Wooten, 2008). This post‐translational modification is important for neurotrophin receptor function since defective ubiquitination of TrkA causes defective endosomal trafficking, impaired degradation and increased recycling of this receptor, which was proposed to lead to the increased survival of sensory neurons in vivo (Yu et al, 2014). Our findings now suggest that BICD1 plays a role in this pathway by regulating the balance between these two degradation fates. We speculate that impaired targeting of TrkB to lysosomes in neurons lacking BICD1 might cause an increased ubiquitination and re‐routing of this receptor to the proteasome and the recycling pathway, resulting in the increased targeting of TrkB and p75NTR to the plasma membrane, possibly via a SNX/retromer‐mediated retrieval route (Fig 8). Such a mechanism would be expected to ensure appropriate TrkB‐mediated signalling output in response to BDNF availability and according to the composition of receptor complexes bound to this ligand.
Several lines of evidence indicate that impairments of the trafficking and/or degradation of neurotrophin receptors alter the output of neurotrophin‐signalling cascades (Reichardt, 2006; Ascano et al, 2012). For example, overexpression of Hrs, a component of the endosomal sorting complex required for transport (ESCRT), in PC12 cells, caused a redirection of TrkB to the recycling route and a more sustained ERK1/2 activation upon BDNF treatment (Huang et al, 2009). Similar behaviour has also been described for TrkA in the same cell line, where inhibition of endosomal maturation by overexpression of a dominant‐negative Rab7 mutant resulted in the accumulation of TrkA‐containing endosomes and an increased ERK1/2 signalling (Saxena et al, 2005). Furthermore, proteosomal inhibition induced a sustained NGF‐dependent ERK1/2 activation (Moises et al, 2009). Based on these results, we investigated the signalling responses to BDNF in Bicd1gt/gt MNs and found that TrkB phosphorylation and the downstream activation of PI3K‐AKT and Ras‐ERK1/2 were decreased in Bicd1gt/gt MNs (Fig 7). Notably, AKT phosphorylation levels, albeit lower, remain stable over a longer period in Bicd1gt/gt MNs compared to wild‐type cells (Fig 7G), supporting the notion that receptor recycling is an important mechanism to sustain signalling by rapidly returning growth factor receptors to the plasma membrane (Chen et al, 2005; Huang et al, 2013).
A likely explanation for the lower signalling capacity of Bicd1gt/gt MNs is the reduced quantity of signalling‐competent TrkB.FL relative to the dominant inhibitory TrkB.T1 isoform present on the surface of BICD1‐depleted neurons (Fig 6C–E). This change in the stoichiometry of TrkB pools combined with the increased levels of p75NTR on the cell surface of Bicd1gt/gt MNs may represent an adaptive response to overstimulation caused by defective degradation of activated TrkB.FL. In this light, BICD1‐depleted MNs could adapt to these unfavourable conditions by altering their cell surface levels of p75NTR and repertoire of TrkB isoforms in order to protect themselves from chronic TrkB.FL‐mediated overstimulation, which is in agreement with the neuronal response to excitotoxic insult during brain ischaemia and traumatic brain injury (Gomes et al, 2012).
In conclusion, we would like to propose a model by which BICD1 controls the trafficking of activated neurotrophin receptors to appropriate degradation routes in order to maintain an optimal response to neurotrophin stimulation. By impinging on this trophic response, BICD1 functions to modulate both the signalling amplitude and duration elicited by ligand‐bound neurotrophin receptors. In this context, BICD1 serves to fine‐tune neurotrophin receptor signalling by maintaining the balance between receptor degradation over recycling so that the receptor pool not destined for degradation is targeted back to the plasma membrane by a retromer‐mediated sorting process (Fig 8). This equilibrium ensures optimal signal intensity and/or duration by delivering the appropriate repertoire of neurotrophin receptors to the plasma membrane where they are available to respond to their cognate ligands, which are especially abundant during neuronal growth. BICD1 function would therefore be expected to be critical during nervous system development when neurons rely on neurotrophins for their specification, differentiation and survival. Indeed, this may be one reason why BICD1 is highly expressed together with neurotrophins and their receptors in the developing nervous system (Fig 1 and Supplementary Fig S1). TrkB‐BDNF‐signalling mechanisms are instrumental in regulating nervous system development and are key modulators of synaptic plasticity in adulthood. Intriguingly, dendritic elongation and branching of cortical neurons is controlled by the differential activation of TrkB.FL versus TrkB.T1 (Yacoubian & Lo, 2000), whilst p75NTR is a negative modulator of dendrite complexity in hippocampal neurons (Zagrebelsky et al, 2005). These functions are very likely to operate in concert with BICD1, which was recently shown to be essential for the correct neuronal patterning and dendritic branching in C. elegans (Aguirre‐Chen et al, 2011). These studies and our current work collectively indicate that BICD1 helps to coordinate the complex membrane sorting of neurotrophin‐signalling endosomes during nervous system development and may regulate the activity of other receptor tyrosine kinases in different cell types.
Materials and Methods
All experiments were carried out following the guidelines of the Cancer Research UK genetic manipulation procedures. Animal work was carried out under licence from the UK Home Office in accordance with the Animals (Scientific Procedures) Act 1986.
Unless stated otherwise, all chemical reagents were supplied by Sigma. Tissue culture media, supplements and AlexaFluor555‐conjugated CTB and secondary antibodies were purchased from Life Technologies.
Actin (AC‐15), BICD1 (HPA041309), BICD2 (HPA024452) and FLAG (M1) were all obtained from Sigma. BDNF (N‐20), LAMP2 (ABL‐93), Rab6 and pan‐Trk (C‐14) were purchased from Santa Cruz. AKT (#9272), phospho‐AKT (S473, #4060), ERK1/2 (#9102), phospho‐ERK1/2 (#9101), TrkB (80E3) and phospho‐TrkB (Y706/707, C50F3) were obtained from Cell Signalling. TrkB antibody (#07‐225, used for live cell internalisation assays) and MAP2 were purchased from Millipore; βIII tubulin (TUJ1) was obtained from Covance; HB9 and SOD1 (ab16831) were obtained from Abcam; Islet1/2 was purchased from Developmental Studies Hybridoma Bank; FK2 was obtained from Enzo Life Sciences; mouse monoclonals to p150Glued and p50 were purchased from BD Biosciences. αp75NTR (5410) was described in (Deinhardt et al, 2007). SNX1 and Vps26 antibodies were a kind gift of Matthew Seaman, University of Cambridge, UK.
A BDNF‐mCherry expression construct was kindly provided by F. Saudou (Institut Curie, Orsay, F; Gauthier et al, 2004) and transfected using Lipofectamine 2000 (Life Technologies) into HEK293‐FT cells, which had previously been adapted to grow in suspension in HyClone Serum‐Free Medium (SFM; Thermo Scientific). SFM conditioned with secreted BDNF‐mCherry was collected after 72 h, centrifuged at high speed and the supernatant concentrated approximately 15‐fold by force filtration using Amicon Ultra Centrifugal filters (Ultracel 30K; Millipore). The concentrate was snap frozen at −80°C and then analysed by western blotting against recombinant BDNF (R&D Systems) to assess BDNF‐mCherry concentration (Deinhardt et al, 2006b) before performing FLAG‐TrkB internalisation experiments in N2A cells.
Bicd1gt/+ chimeric embryos and lacZ expression analysis
10–15 RRP227 ES cells were injected into blastocyst stage embryos collected from superovulated C57BL/6J female mice that had been mated to C57BL/6J male mice. Embryos were transferred to pseudo‐pregnant (2.5 days post‐coitum) recipient mice according to standard protocols (Nagy et al, 2003). To assess Bicd1‐lacZ expression patterns during mouse development, E11.5‐E14.5 embryos were fixed at room temperature for 15–30 min in 0.1 M sodium phosphate, pH 7.3, 0.4% paraformaldehyde (PFA), 5 mM EGTA, 2 mM MgCl2, washed three times in 0.1 M sodium phosphate pH 7.3, 2 mM MgCl2, 0.1% sodium deoxycholate, 0.02% NP‐40 and finally transferred into developing solution (100 mM sodium phosphate, pH 7.3, 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% NP‐40, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 1 mg/ml X‐gal (Roche), for 30 min to 5 h at 37°C or overnight in the dark. The reaction was quenched by rinsing several times in PBS, and embryos were then post‐fixed in 4% PFA and stored in 70% ethanol prior to imaging. After imaging, some samples were paraffin embedded, sectioned, DAB immunostained and counterstained with Nile red.
Embryonic stem‐cell‐derived MNs
Embryonic stem cells were grown on fish skin gelatin‐coated flasks in Glasgow Minimal Essential Medium (GMEM), 5% ES cell‐qualified foetal bovine serum FBS, 5% knockout serum replacement (KSR), 1% GLUTAMAX, 0.1 mM 2‐mercaptoethanol and 1,000 units/ml of leukaemia inhibitory factor (ESGRO, Millipore). To generate MNs, 1.5 × 106 ES cells were grown in suspension on a 10‐cm non‐tissue culture‐treated Petri dish containing differentiation (DFNK) medium: 45% neurobasal, 45% DMEM/Ham's‐F12, 10% KSR, 1% GLUTAMAX and 0.1 mM 2‐mercaptoethanol. The following day, embryoid bodies (EBs) were gently centrifuged and re‐suspended in 10 ml of fresh DFNK medium and plated on a new Petri dish. The following day, the greatly enlarged EBs were allowed to sediment by gravity and re‐suspended in fresh DFNK medium supplemented with 1 μM all‐trans retinoic acid (RA) and 333 nM Smoothened Agonist (SAG; Enzo Life Sciences). EBs were maintained under these conditions for a further 4 days (medium changed every other day) and then dissociated with 0.025% porcine pancreatic trypsin in 1 ml PBS for 7 min at 37°C and processed as described previously for the dissociation of mouse E13.5 spinal cord MNs (Hafezparast et al, 2003). Cells were plated onto poly‐D‐ornithine and laminin‐coated dishes in MN growth medium: neurobasal medium supplemented with 2% B27, 2% heat‐inactivated horse serum, 1% GLUTAMAX, 25 μM 2‐mercaptoethanol, 10 ng/ml rat ciliary neurotrophic factor (CNTF; R&D Systems), 100 pg/ml rat glial cell line‐derived neurotrophic factor (GDNF; R&D Systems) and 1 μM RA.
Generation of homozygous RRP227 ES cells
Mouse ES cells with a gene trap insertion in the first intron of Bicd1 (RRP227; http://www.informatics.jax.org/javawi2/servlet/WIFetch?page=alleleDetailandkey=544886) were obtained from the Mutant Mouse Regional Resource Center. Homozygous Bicd1gt/gt cells were generated as previously described (Lefebvre et al, 2001). Briefly, heterozygous cells were seeded at 30% confluence and maintained under standard feeder‐free ES cell culture conditions in medium containing 1.5 mg/ml G418 until distinct clones of antibiotic‐resistant cells appeared. Several clones were picked up into a drop of 0.025% trypsin solution and seeded into separate wells of a 24‐well plate and grown in the presence of 500 ng/ml G418. Loss of heterozygosity was assessed by semi‐quantitative real‐time PCR using the One‐Step RT‐PCR kit (Life Technologies) and primers specific for the wild‐type Bicd1 cDNA (forward: ggc tgg tgg tgc tgg agg aga a; reverse: gtg gac act agt ttc tgc aat gtg a).
The G418‐resistant ES cell clone that showed the most marked reduction in PCR product relative to the heterozygous parent cell line was then selected for further quantification by quantitative real‐time PCR, which confirmed an approximately 70% reduction in Bicd1 expression relative to wild‐type ES cells.
Quantitative real‐time PCR
Total RNA was extracted from ES cell‐derived MN cultures 4–5 days after the plating of disaggregated EBs, using either Trizol (Life Technologies) or RNeasy kits (Qiagen). One to 2 μg of total RNA was used to synthesise cDNA using the Superscript‐VILO cDNA synthesis kit (Life Technologies). cDNA was diluted tenfold, and PCR amplified on a 7500 Fast Real‐Time PCR (Applied Biosystems) using intron spanning primers (Supplementary Table S2) and EXPRESS SYBR Green ER master mix (Life Technologies).
BICD1‐GFP and FLAG‐TrkB overexpression in N2A cells
N2A cells were transfected with FLAG‐TrkB constructs (kindly provided by Francis Lee, Weill Cornell Medical College, NY, USA) and BICD1‐GFP using Lipofectamine 2000 according to the manufacturer's instructions. Approximately 13 h after transfection, antibody uptake experiments were performed on N2A cells by incubation with FLAG‐tag antibody (clone M1, 1:1000) in the presence either of recombinant BDNF (100 ng/ml) or purified mCherry‐BDNF for 1 h at 37°C. Cells were then acid‐washed for 2 min, washed in PBS and fixed with 4% PFA for 20 min. Cells were then immunostained with AlexaFluor555‐ or AlexaFluor488‐conjugated anti‐mouse antibodies to detect internalised FLAG‐TrkB and primary antibodies targeting endogenous SNX1 or BICD1.
Immunofluorescence and immunohistochemistry
Motor neurons were seeded onto poly‐D‐ornithine and laminin‐coated coverslips, maintained under standard culture conditions for 4–5 days before fixation with 4% PFA for 15 min at room temperature. Fixed cells were washed and blocked in 2% bovine serum albumin (BSA) in PBS with or without 0.2% Triton X‐100 for 20 min at room temperature prior to incubation for 1 h with primary antibodies (see above) followed by AlexaFluor‐conjugated secondary antibodies (1:500). Samples were then counterstained with DRAQ5 (Biostatus), post‐fixed with PFA and mounted with Mowiol.
E11.5–14.5 embryos were fixed in neutral‐buffered formalin overnight, processed for paraffin embedding and then sectioned. Sections (4 μm) were microwaved for 15 min in 0.01 M sodium citrate buffer, pH 6.0 for antigen retrieval, blocked with 10% normal donkey serum, 1% BSA and stained overnight at 4°C with a combination of mouse anti‐βIII tubulin and either rabbit anti‐BDNF (N‐20), rabbit anti‐pan‐Trk (C‐14) or αp75NTR (5410). Following extensive washing in PBS, sections were stained with biotinylated horse anti‐rabbit IgG (Vector Labs) and AlexaFluor488‐conjugated donkey anti‐mouse IgG for 1 h. After washing, sections were incubated in AlexaFluor555‐conjugated streptavidin (Life Technologies) for 45 min at room temperature, washed, incubated for 30 min in 0.1% Sudan Black dissolved in 70% ethanol to quench auto‐fluorescence and then mounted in Hardset Mount containing DAPI (Vector). The same protocol was used for horseradish peroxidase (HRP) immunohistochemistry except that sections were firstly quenched with 1.6% H2O2/PBS before blocking and staining with a single primary antibody, followed by biotinylated secondary antibody and ABC reagent (Vector Labs), developed with DAB (Vector Labs) and counterstained with haematoxylin.
Cell surface biotinylation
The pool of neurotrophin receptors on the plasma membrane was retrieved using a cell surface biotinylation kit (Thermo Scientific) according to the manufacturer's instructions. Briefly, wild‐type and Bicd1gt/gt MNs were assayed under steady‐state conditions 4–5 days after plating at high density onto 6‐cm dishes, by cooling on wet ice before removing the growth medium, washing with ice‐cold PBS and crosslinking cell‐surface‐exposed proteins with sulfo‐NHS‐SS‐biotin for 30 min on ice. After quenching, cells were scraped, pelleted, washed twice with ice‐cold Tris‐buffered saline (TBS) and lysed on ice for 30 min. Insoluble proteins were pelleted by centrifugation and supernatants adjusted to the same protein concentration. Biotinylated proteins from 100 μg of total lysate were isolated with neutravidin agarose beads prior to western blot analysis.
Endosome isolation and Western blotting
MION‐conjugated HCT was used to purify HCT‐positive endosomes, which were isolated as previously described (Wade et al, 2012). SDS‐PAGE was performed using 4–12% NuPAGE Bis‐Tris gradient gels (Life Technologies) according to the manufacturer's instructions and blotted onto polyvinylidene fluoride (PVDF) membranes. Membranes were blocked in 5% skimmed milk or 5% BSA dissolved in TBS containing 0.05% Tween‐20 (TBST) for 1 h at room temperature and then incubated with primary antibodies diluted 1:1,000 or 1:2,000 in TBST for 1 h at room temperature or overnight at 4°C. Blots were then washed and incubated with appropriate HRP‐conjugated secondary antibodies (GE Healthcare). Immunoreactivity was detected using Luminata or Crescendo ECL substrates (Millipore) and ECL‐Hyperfilm (GE Healthcare).
Internalisation assays for HCT, αp75NTR and αTrkB (Upstate, 1:1,000) were performed on wild‐type and Bicd1gt/gt motor neurons seeded onto coverslips. Experiments designed to rescue the Bicd1 depletion phenotypes were carried out on Bicd1gt/gt motor neurons transfected with a full length mouse Bicd1 cDNA (MGC‐27566, LGC Promochem) cloned into EcoR1/BamH1 sites of EGFP‐N1 (Clontech). This BICD1‐GFP construct (see Terenzio et al., 2014) was transfected into motor neurons by Magnetofection using Neuromag (OZ Biosciences) following manufacturer's specifications. Cells were assayed within 16 h of transfection.
HCT and αp75NTR and αTrkB internalisation/accumulation assays were performed as described in the main text, but in the presence of lysosomal inhibitors (leupeptin 200 μM, E64D 2 μM, pepstatin A 20 μM) or an equivalent volume of DMSO. Cells were allowed to internalise the probes for 1 h at 37°C, acid‐washed for 2 min, washed in PBS and fixed with 4% PFA for 20 min. To detect internalised αp75NTR or αTrkB, the cells were immunostained with anti‐rabbit AlexaFluor555‐conjugated IgG.
For biochemical assessments of internalised receptor fate, a pulse‐chase antibody feeding assay was performed as follows: MN cultures were incubated with αTrkB and BDNF in the presence or absence of lysosomal inhibitors in complete growth medium as described above, but instead of a 1‐h continuous feed, a 15‐min internalisation pulse was performed. This was followed by washing the cells three times with fresh antibody‐free medium and incubation for a further 1 or 2 h as above, but in the absence of αTrkB. To assess TrkB ubiquitination, the same protocol was used with the addition of MG132 (10 μM), which was present throughout the pulse and chase periods. The quantity of initially internalised αTrkB/TrkB complex (15 min pulse) and non‐degraded αTrkB/TrkB remaining after the chase periods was determined by immunoprecipitating the antibody/receptor complex from cell lysates prepared as described below for the signalling assays. Lysates were then incubated for 1 h at 4°C with 10 μl of pre‐washed protein‐G coated Dynabeads (Life Technologies) per sample. TrkB‐bound beads were then washed three times in lysis buffer and captured antibodies eluted with Laemmlli sample buffer followed by SDS‐PAGE and western blotting. The top half of these blots was first probed using the rabbit anti‐TrkB (80E3) antibody. Some blots were then stripped and re‐probed for ubiquitin using the mouse FK2 antibody. The lower half of these same blots were probed separately with HRP‐conjugated anti‐rabbit immunoglobulins in order to detect the immunoglobulin chains of internalised αTrkB.
Cholera toxin B subunit accumulation assay
Wild‐type and Bicd1gt/gt MNs were allowed to internalise AlexaFluor555‐CTB (1 μg/ml) for 1 h at 37°C, acid‐washed for 2 min, washed in PBS and fixed with 4% PFA for 20 min. Samples were immunostained for βIII tubulin and imaged. The amount of CTB accumulation in the Golgi area was quantified using ImageJ.
Neurite outgrowth assay
Wild‐type and Bicd1gt/gt MNs were plated at equal density, fixed 48 h after plating with 4% PFA for 20 min, immunostained for βIII tubulin and imaged. Quantification of total outgrowth, maximum process length and number of branches was performed using Metamorph (Molecular Devices).
Wild‐type and Bicd1gt/gt MNs were starved for 5 h at 37°C in neurobasal medium and then stimulated with 100 ng/ml of BDNF in the same medium. At the appropriate time points, cells were placed on ice immediately after removing the growth medium and then lysed in 10 mM Tris‐HCl pH 8.0, 150 mM NaCl, 1% NP‐40, 1 mM EDTA containing HALT protease and phosphatase inhibitors (Thermo Scientific) for 30 min. Insoluble proteins were pelleted and protein concentration assayed in the supernatants before addition of sample buffer. Samples were heated for 10 min at 70°C and 5–15 μg of protein per sample loaded in SDS‐PAGE prior to western blotting for pTrkB, phospho‐AKT and pERK1/2. Phosphorylation levels were quantified either by re‐probing the same membranes, or in some cases, by running the same lysates in parallel and immunoblotting for the respective total proteins.
Axonal retrograde transport assay
Axonal retrograde transport kinetics of HCT and αp75NTR in wild‐type and in Bicd1gt/gt MNs was performed as previously described (Lalli & Schiavo, 2002; Deinhardt et al, 2006b) and quantified using Motion Analysis software (Kinetic Imaging; Bohnert & Schiavo, 2005; Deinhardt et al, 2006b).
Transmission electron microscopy
HCT was conjugated to colloidal gold by mixing 250 μg of purified HCT in 0.5 ml of 2 mM sodium tetraborate to 1 ml of colloidal gold particles (10 nm; British Biocell) previously adjusted to pH 6.0. αp75NTR (#5411; 200 μg in 0.2 ml) and αTrkB (#07‐225; 60 μg in 0.2 ml) were dialysed against 2 mM sodium tetraborate and incubated with 2 ml (5 nm) and 0.2 ml (20 nm), respectively, of colloidal gold previously adjusted to pH 9.0. Samples were stirred at room temperature for 10 min. BSA was added to a final concentration of 1%, and the mixture stirred for a further 10 min. The suspension was finally pelleted at 45,000 g for 45 min (αp75NTR), 30 min (HCT) and 10 min (αTrkB), resuspended in the original volume of 20 mM Tris‐NaOH pH 8.2, 150 mM NaCl, 1% BSA and stored at 4°C for a maximum of 2–3 weeks.
Motor neurons plated on coverslips were incubated with 20 nM nanogold‐conjugated HCT for 2 h at 37°C prior to washing. Cells were fixed in 2.5% glutaraldehyde, 4% PFA in Sorensen's phosphate buffer at room temperature for 20 min, post‐fixed in osmium tetroxide, stained with tannic acid and dehydrated progressively up to 100% ethanol. Finally, coverslips were embedded in an Epon epoxy resin, sectioned (70–75 nm) and stained with lead citrate. Images were acquired using a Tecnai Spirit Biotwin (FEI) transmission electron microscope. Random grids were visually scanned for the presence of nanogold‐containing organelles by two independent operators and classified as described in the main text.
ImageJ was used for the quantification of western blots and immunofluorescence staining in all experiments. ImageJ was used to threshold the fluorescence staining of interest and quantify the thresholded voxels. Immunostaining for neuronal‐specific proteins, such as βΙΙΙ tubulin, was used as object masks to quantify both immunostaining intensity as well as fluorescent probe binding and internalisation.
M.T. designed, optimised and performed the siRNA screen, validated the resulting hits and analysed and interpreted the data. M.G. performed embryo analysis, biotinylation assays, antibody capture and signalling experiments and interpreted the data. B. S‐D. carried out immunohistochemistry and tissue analyses. I.R. generated embryos from Bicd1gt/+ ES cells. M.G., M.T. and K.W. contributed to the remaining work. G.S. designed the study, contributed to data analyses and interpretation. D.I‐H. provided critical comments and contributed to revision of the text. M.G., M.T. and G.S. wrote the manuscript and assembled the figures.
Conflict of interest
The authors declare that they have no conflict of interest.
Supplementary Figure S1
Supplementary Figure S2
Supplementary Figure S3
Supplementary Figure S4
Supplementary Figure S5
Supplementary Figure S6
Supplementary Figure S7
Supplementary Figure S8
Supplementary Table S1
Supplementary Table S2
We thank Michael Parkinson and Ken Blight for help with electron microscopy sample preparation and analyses, Frederic Saudou for BDNF‐mCherry expression vector, Francis Lee for the FLAG‐TrkB construct, Matthew Seaman for anti‐SNX1 and Vps26 antibodies and members of the Molecular Neuropathobiology Laboratory for constructive comments. This work was supported by Cancer Research UK, the Marie Curie Actions Human Resources and Mobility Training Network ‘Endocyte’ (MRTN‐CT‐2006‐035528; to MT), the FP7‐HEALTH‐2007‐B grant ‘BrainCAV’ (FP7‐222992; to GS) and Weizmann‐UK ‘Making Connections’ grant (to GS).
FundingCancer Research UK
- © 2014 The Authors