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Retrograde transport of TrkB-containing autophagosomes via the adaptor AP-2 mediates neuronal complexity and prevents neurodegeneration

View Article: PubMed Central - PubMed

ABSTRACT

Autophagosomes primarily mediate turnover of cytoplasmic proteins or organelles to provide nutrients and eliminate damaged proteins. In neurons, autophagosomes form in distal axons and are trafficked retrogradely to fuse with lysosomes in the soma. Although defective neuronal autophagy is associated with neurodegeneration, the function of neuronal autophagosomes remains incompletely understood. We show that in neurons, autophagosomes promote neuronal complexity and prevent neurodegeneration in vivo via retrograde transport of brain-derived neurotrophic factor (BDNF)-activated TrkB receptors. p150Glued/dynactin-dependent transport of TrkB-containing autophagosomes requires their association with the endocytic adaptor AP-2, an essential protein complex previously thought to function exclusively in clathrin-mediated endocytosis. These data highlight a novel non-canonical function of AP-2 in retrograde transport of BDNF/TrkB-containing autophagosomes in neurons and reveal a causative link between autophagy and BDNF/TrkB signalling.

No MeSH data available.


Direct binding of AP-2α to LC3b and association with p150Glued/dynactin.(a) Upper panel: purified recombinant LC3b-His6 detected by immunoblotting directly binds to GST-AP-2αΑ with preference (about threefold) over GST-AP-2αC. Input, 2% of the total recombinant LC3b added to the assay. Lower panel: ponceau-stained membrane. Example from n=3 independent experiments. (b) Complex formation of neuronal AP-2 with LC3b and the p150Glued subunit of dynactin. Affinity purifications of mouse brain lysates using GST-LC3b as bait co-purified AP-2αA and, to a lesser extent AP-2αC, as well as p150Glued, but not the negative control protein Akt. Input, 1.5% of lysate added to the assay. Representative example from n=4 independent experiments. (c) Co-immunoprecipitation of endogenous AP-2 with p150Glued, but not with Kif5A from rat brain lysate using antibodies against AP-2β. Hsc70, used as a negative control, was not co-precipitated. Input, 10% of lysate added to the assay. Representative example from n=3 independent experiments. (d) AP-2β-ear interacts with p150Glued. AviGFP-tagged p150Glued or β-galactosidase were expressed together with BirA in HEK293T cells, affinity-purified using M-280 streptavidin Dynabeads, incubated with recombinant His6-AP-2β-ear and analysed by immunoblotting. Input, 10% of lysate added to the assay. Representative example from n=3 independent experiments. (e) AP-2 interacts with LC3b and p150Glued. Affinity purifications using a GST-tagged N-terminal fragment of Stonin 2 as bait co-purified endogenous AP-2 (detected via its μ and α subunits), LC3b and p150Glued. Input, 1.4% of lysate added to the assay. Representative example from n=3 independent experiments.
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f2: Direct binding of AP-2α to LC3b and association with p150Glued/dynactin.(a) Upper panel: purified recombinant LC3b-His6 detected by immunoblotting directly binds to GST-AP-2αΑ with preference (about threefold) over GST-AP-2αC. Input, 2% of the total recombinant LC3b added to the assay. Lower panel: ponceau-stained membrane. Example from n=3 independent experiments. (b) Complex formation of neuronal AP-2 with LC3b and the p150Glued subunit of dynactin. Affinity purifications of mouse brain lysates using GST-LC3b as bait co-purified AP-2αA and, to a lesser extent AP-2αC, as well as p150Glued, but not the negative control protein Akt. Input, 1.5% of lysate added to the assay. Representative example from n=4 independent experiments. (c) Co-immunoprecipitation of endogenous AP-2 with p150Glued, but not with Kif5A from rat brain lysate using antibodies against AP-2β. Hsc70, used as a negative control, was not co-precipitated. Input, 10% of lysate added to the assay. Representative example from n=3 independent experiments. (d) AP-2β-ear interacts with p150Glued. AviGFP-tagged p150Glued or β-galactosidase were expressed together with BirA in HEK293T cells, affinity-purified using M-280 streptavidin Dynabeads, incubated with recombinant His6-AP-2β-ear and analysed by immunoblotting. Input, 10% of lysate added to the assay. Representative example from n=3 independent experiments. (e) AP-2 interacts with LC3b and p150Glued. Affinity purifications using a GST-tagged N-terminal fragment of Stonin 2 as bait co-purified endogenous AP-2 (detected via its μ and α subunits), LC3b and p150Glued. Input, 1.4% of lysate added to the assay. Representative example from n=3 independent experiments.

Mentions: The co-trafficking of AP-2 with LC3 on autophagosomes raises the question how neuronal AP-2 is recruited to these carriers. While the β, μ and σ subunits of AP-2 complex are made from a single gene in mammals, the α subunit is encoded by two isogenes termed αA and αC that undergo alternative splicing in the brain. Recent data suggest that AP-2 can associate with LC3 via a putative LIR motif within the appendage domain of the AP-2αA subunit29. To probe whether AP-2 via its α-appendage domain directly binds to LC3 we carried out binding assays using purified proteins (Fig. 2a, Supplementary Fig. 2a,c). Purified recombinant LC3 was found to bind to the GST-tagged appendage domains of both AP-2α and AP-2αC with a preference for AP-2αA over AP-2αC (Fig. 2a). In line with this, endogenous AP-2αA/C co-purified with GST-LC3b (Supplementary Fig. 2c) in affinity chromatography experiments from brain lysates (Fig. 2b), while, conversely, GST-AP-2αA and, less well, GST-AP-2αC associated with native LC3b (Supplementary Fig. 2b). Consistent with the preferential retrograde transport of AP-2-containing LC3-positive autophagosomes, we found endogenous AP-2 to co-immunoprecipitate with the p150Glued subunit of dynactin, a cofactor for the retrograde microtubule-based motor dynein, but not with the anterograde trafficking motor Kif5A from detergent-extracted rat brain lysates (Fig. 2c). Moreover, p150Glued expressed in HEK293T cells was able to capture the purified appendage domain of AP-2β in binding assays (Fig. 2d). Finally, affinity purification of endogenous AP-2 using GST-fused Stonin 2 as a bait resulted in the co-purification of both LC3b and p150Glued (Fig. 2e, see also Supplementary Fig. 2d), suggesting that all three proteins act as part of a complex. Consistent with these biochemical data we observed the close colocalization of endogenous LC3b with p150Glued and AP-2 in neurons treated with folimycin (Supplementary Fig. 2e–h). Collectively, these findings indicate that AP-2 associates with LC3 and p150Glued in a protein complex that may mediate retrograde transport of autophagosomes.


Retrograde transport of TrkB-containing autophagosomes via the adaptor AP-2 mediates neuronal complexity and prevents neurodegeneration
Direct binding of AP-2α to LC3b and association with p150Glued/dynactin.(a) Upper panel: purified recombinant LC3b-His6 detected by immunoblotting directly binds to GST-AP-2αΑ with preference (about threefold) over GST-AP-2αC. Input, 2% of the total recombinant LC3b added to the assay. Lower panel: ponceau-stained membrane. Example from n=3 independent experiments. (b) Complex formation of neuronal AP-2 with LC3b and the p150Glued subunit of dynactin. Affinity purifications of mouse brain lysates using GST-LC3b as bait co-purified AP-2αA and, to a lesser extent AP-2αC, as well as p150Glued, but not the negative control protein Akt. Input, 1.5% of lysate added to the assay. Representative example from n=4 independent experiments. (c) Co-immunoprecipitation of endogenous AP-2 with p150Glued, but not with Kif5A from rat brain lysate using antibodies against AP-2β. Hsc70, used as a negative control, was not co-precipitated. Input, 10% of lysate added to the assay. Representative example from n=3 independent experiments. (d) AP-2β-ear interacts with p150Glued. AviGFP-tagged p150Glued or β-galactosidase were expressed together with BirA in HEK293T cells, affinity-purified using M-280 streptavidin Dynabeads, incubated with recombinant His6-AP-2β-ear and analysed by immunoblotting. Input, 10% of lysate added to the assay. Representative example from n=3 independent experiments. (e) AP-2 interacts with LC3b and p150Glued. Affinity purifications using a GST-tagged N-terminal fragment of Stonin 2 as bait co-purified endogenous AP-2 (detected via its μ and α subunits), LC3b and p150Glued. Input, 1.4% of lysate added to the assay. Representative example from n=3 independent experiments.
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f2: Direct binding of AP-2α to LC3b and association with p150Glued/dynactin.(a) Upper panel: purified recombinant LC3b-His6 detected by immunoblotting directly binds to GST-AP-2αΑ with preference (about threefold) over GST-AP-2αC. Input, 2% of the total recombinant LC3b added to the assay. Lower panel: ponceau-stained membrane. Example from n=3 independent experiments. (b) Complex formation of neuronal AP-2 with LC3b and the p150Glued subunit of dynactin. Affinity purifications of mouse brain lysates using GST-LC3b as bait co-purified AP-2αA and, to a lesser extent AP-2αC, as well as p150Glued, but not the negative control protein Akt. Input, 1.5% of lysate added to the assay. Representative example from n=4 independent experiments. (c) Co-immunoprecipitation of endogenous AP-2 with p150Glued, but not with Kif5A from rat brain lysate using antibodies against AP-2β. Hsc70, used as a negative control, was not co-precipitated. Input, 10% of lysate added to the assay. Representative example from n=3 independent experiments. (d) AP-2β-ear interacts with p150Glued. AviGFP-tagged p150Glued or β-galactosidase were expressed together with BirA in HEK293T cells, affinity-purified using M-280 streptavidin Dynabeads, incubated with recombinant His6-AP-2β-ear and analysed by immunoblotting. Input, 10% of lysate added to the assay. Representative example from n=3 independent experiments. (e) AP-2 interacts with LC3b and p150Glued. Affinity purifications using a GST-tagged N-terminal fragment of Stonin 2 as bait co-purified endogenous AP-2 (detected via its μ and α subunits), LC3b and p150Glued. Input, 1.4% of lysate added to the assay. Representative example from n=3 independent experiments.
Mentions: The co-trafficking of AP-2 with LC3 on autophagosomes raises the question how neuronal AP-2 is recruited to these carriers. While the β, μ and σ subunits of AP-2 complex are made from a single gene in mammals, the α subunit is encoded by two isogenes termed αA and αC that undergo alternative splicing in the brain. Recent data suggest that AP-2 can associate with LC3 via a putative LIR motif within the appendage domain of the AP-2αA subunit29. To probe whether AP-2 via its α-appendage domain directly binds to LC3 we carried out binding assays using purified proteins (Fig. 2a, Supplementary Fig. 2a,c). Purified recombinant LC3 was found to bind to the GST-tagged appendage domains of both AP-2α and AP-2αC with a preference for AP-2αA over AP-2αC (Fig. 2a). In line with this, endogenous AP-2αA/C co-purified with GST-LC3b (Supplementary Fig. 2c) in affinity chromatography experiments from brain lysates (Fig. 2b), while, conversely, GST-AP-2αA and, less well, GST-AP-2αC associated with native LC3b (Supplementary Fig. 2b). Consistent with the preferential retrograde transport of AP-2-containing LC3-positive autophagosomes, we found endogenous AP-2 to co-immunoprecipitate with the p150Glued subunit of dynactin, a cofactor for the retrograde microtubule-based motor dynein, but not with the anterograde trafficking motor Kif5A from detergent-extracted rat brain lysates (Fig. 2c). Moreover, p150Glued expressed in HEK293T cells was able to capture the purified appendage domain of AP-2β in binding assays (Fig. 2d). Finally, affinity purification of endogenous AP-2 using GST-fused Stonin 2 as a bait resulted in the co-purification of both LC3b and p150Glued (Fig. 2e, see also Supplementary Fig. 2d), suggesting that all three proteins act as part of a complex. Consistent with these biochemical data we observed the close colocalization of endogenous LC3b with p150Glued and AP-2 in neurons treated with folimycin (Supplementary Fig. 2e–h). Collectively, these findings indicate that AP-2 associates with LC3 and p150Glued in a protein complex that may mediate retrograde transport of autophagosomes.

View Article: PubMed Central - PubMed

ABSTRACT

Autophagosomes primarily mediate turnover of cytoplasmic proteins or organelles to provide nutrients and eliminate damaged proteins. In neurons, autophagosomes form in distal axons and are trafficked retrogradely to fuse with lysosomes in the soma. Although defective neuronal autophagy is associated with neurodegeneration, the function of neuronal autophagosomes remains incompletely understood. We show that in neurons, autophagosomes promote neuronal complexity and prevent neurodegeneration in vivo via retrograde transport of brain-derived neurotrophic factor (BDNF)-activated TrkB receptors. p150Glued/dynactin-dependent transport of TrkB-containing autophagosomes requires their association with the endocytic adaptor AP-2, an essential protein complex previously thought to function exclusively in clathrin-mediated endocytosis. These data highlight a novel non-canonical function of AP-2 in retrograde transport of BDNF/TrkB-containing autophagosomes in neurons and reveal a causative link between autophagy and BDNF/TrkB signalling.

No MeSH data available.