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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.

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Related in: MedlinePlus

AP-2 regulates autophagosome transport in neurons.(a) Time-lapse images of mRFP-LC3-positive puncta (arrows) in WT and AP-2μ KO neurons. Scale bar, 5 μm. (b) Kymographs of mRFP-LC3 carriers generated from a. (c) Average retrograde velocity of mRFP-LC3 carriers in WT and AP-2μ KO neurons. Loss of AP-2μ significantly decreased the LC3 velocity compared to WT controls (WT: 0.44±0.07 μm s−1, KO: 0.21±0.03 μm s−1, *P=0.019, n=4 independent experiments, 49–67 neurites per condition). (d–e) Electron micrographs of synapses from cultured WT and AP-2μ KO neurons. AP-2 KO neurons accumulate dense vesicular bodies with the majority representing concentric multilamellar structures (black boxes in d represent magnified areas in e). Scale bars, (d) 500 nm, (e) 100 nm. Sp, spine. See also Supplementary Fig. 3l–o. (f) Percentage of WT and KO synapses containing dense vesicular bodies (WT: 6.00%±1.54%, AP-2 KO: 13.00%±2.52%, *P=0.045, n=4, 100 synapses per condition). (g–i) Representative confocal images of cultured WT and AP-2 KO neurons immunostained for LC3b and Rab7 (white boxes in g represent the magnified areas in h,i). Scale bars, (g) 15 μm, (h,i) 2 μm. (j,k) Accumulation of LC3b-containing structures (LC3b puncta μm−2 are depicted, WT: 0.009±0.002, AP-2 KO: 0.040±0.009, *P=0.016, n=4, in total 39 AP-2 KO and 33 WT neurons) (j) and Rab7-containing structures (Rab7 puncta μm−2 are depicted, WT: 0.004±0.000, AP-2 KO: 0.007±0.000, *P=0.042, n=3, in total 29 AP-2 KO and 23 WT neurons) (k) in AP-2μ-KO neurons. (l) Enhanced colocalization of LC3b with Rab7 on neuronal autophagosomes in absence of AP-2μ based on Pearson's coefficient (Rp) (WT: 0.52±0.04, AP-2 KO: 0.64±0.01, *P=0.032). Rp was calculated for 64–84 regions of interest (ROI) per condition from three independent experiments (n=3). (m,n) Bar diagrams indicating similar numbers of LC3b- (WT: 0.09±0.01, AP-2 KO: 0.1±0.02) (m) and Rab7-positive puncta μm−2 (WT: 0.05±0.02%, AP-2 KO: 0.04±0.01) (n) in WT and AP-2μ KO neurons treated with folimycin.. Shown is the number of puncta per μm2 (n=3 independent experiments, 26 neurons per condition). Data in c,f,j–n are illustrated as box plots as described in Methods. Data reported in the text are mean±s.e.m. NS, non-significant.
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f3: AP-2 regulates autophagosome transport in neurons.(a) Time-lapse images of mRFP-LC3-positive puncta (arrows) in WT and AP-2μ KO neurons. Scale bar, 5 μm. (b) Kymographs of mRFP-LC3 carriers generated from a. (c) Average retrograde velocity of mRFP-LC3 carriers in WT and AP-2μ KO neurons. Loss of AP-2μ significantly decreased the LC3 velocity compared to WT controls (WT: 0.44±0.07 μm s−1, KO: 0.21±0.03 μm s−1, *P=0.019, n=4 independent experiments, 49–67 neurites per condition). (d–e) Electron micrographs of synapses from cultured WT and AP-2μ KO neurons. AP-2 KO neurons accumulate dense vesicular bodies with the majority representing concentric multilamellar structures (black boxes in d represent magnified areas in e). Scale bars, (d) 500 nm, (e) 100 nm. Sp, spine. See also Supplementary Fig. 3l–o. (f) Percentage of WT and KO synapses containing dense vesicular bodies (WT: 6.00%±1.54%, AP-2 KO: 13.00%±2.52%, *P=0.045, n=4, 100 synapses per condition). (g–i) Representative confocal images of cultured WT and AP-2 KO neurons immunostained for LC3b and Rab7 (white boxes in g represent the magnified areas in h,i). Scale bars, (g) 15 μm, (h,i) 2 μm. (j,k) Accumulation of LC3b-containing structures (LC3b puncta μm−2 are depicted, WT: 0.009±0.002, AP-2 KO: 0.040±0.009, *P=0.016, n=4, in total 39 AP-2 KO and 33 WT neurons) (j) and Rab7-containing structures (Rab7 puncta μm−2 are depicted, WT: 0.004±0.000, AP-2 KO: 0.007±0.000, *P=0.042, n=3, in total 29 AP-2 KO and 23 WT neurons) (k) in AP-2μ-KO neurons. (l) Enhanced colocalization of LC3b with Rab7 on neuronal autophagosomes in absence of AP-2μ based on Pearson's coefficient (Rp) (WT: 0.52±0.04, AP-2 KO: 0.64±0.01, *P=0.032). Rp was calculated for 64–84 regions of interest (ROI) per condition from three independent experiments (n=3). (m,n) Bar diagrams indicating similar numbers of LC3b- (WT: 0.09±0.01, AP-2 KO: 0.1±0.02) (m) and Rab7-positive puncta μm−2 (WT: 0.05±0.02%, AP-2 KO: 0.04±0.01) (n) in WT and AP-2μ KO neurons treated with folimycin.. Shown is the number of puncta per μm2 (n=3 independent experiments, 26 neurons per condition). Data in c,f,j–n are illustrated as box plots as described in Methods. Data reported in the text are mean±s.e.m. NS, non-significant.

Mentions: We then monitored the transport of autophagosomes in wild-type (WT) and AP-2μ KO neurons expressing mRFP-eGFP-LC3 by live imaging. In WT neurons mRFP-labelled autophagosomes displayed bidirectional movements with an average retrograde velocity of about 0.4–0.5 μm s−1 (Fig. 3a–c), similar to the values obtained for AP-2μ-mRFP with which it colocalizes (compare Fig. 1) and consistent with earlier data1032. AP-2μ deletion greatly reduced retrograde autophagosome velocity and the mobile fraction of retrograde LC3b-positive carriers (Fig. 3a–c, Supplementary Fig. 3e), while the fraction of stationary LC3b puncta was increased (Supplementary Fig. 3f). In agreement with the function of dynein motors in slow anterograde movement, lack of neuronal AP-2 caused a mild, yet statistically insignificant reduction in anterograde autophagosome transport (Supplementary Fig. 3e,g)33. Transport of mitochondria proceeded unperturbed in absence of neuronal AP-2 (Supplementary Fig. 3h–k). These data suggest that AP-2 regulates retrograde transport of autophagosomes from neurites to the cell soma, where most lysosomes are located510. Consistent with this hypothesis and with our live imaging results analysis of AP-2μ KO neurons by thin-section electron microcopy revealed an accumulation of dense vesicular and concentric multilamellar organelles resembling late-stage autophagosomes (also termed amphisomes) post-fusion with late endosomes (Fig. 3d–f, Supplementary Fig. 3o and below). As reported earlier26, AP-2μ KO neurons displayed a reduced number of synaptic vesicles (to about 60% of those seen in WT), in agreement with its canonical function in synaptic vesicle reformation. The ‘spheroid-like' accumulation of late-stage autophagosomes in neurites and in the soma of AP-2μ KO compared to WT control neurons was confirmed by immunostaining with antibodies against endogenous LC3b and late endosomal Rab7 (Fig. 3g–l for quantifications), indicating that the autophagosomal structures observed by light and electron microscopy have undergone fusion with late endosomes. In contrast, no significant alterations in the number or localization of early endosomes marked by Rab5 (Supplementary Fig. 4a,b) or LAMP1-positive late endosomes/lysosomes (Supplementary Fig. 3l–n, Supplementary Fig. 4c,d) were observed. To probe whether loss of AP-2 may alter LC3b synthesis and/or degradation3435, we treated WT and KO neurons with the lysosomotropic agent folimycin. Folimycin application caused the accumulation of LC3b-positive puncta in WT neurons to reach levels similar to those seen in neurons lacking AP-2μ (Fig. 3m, Supplementary Fig. 4e). Thus, LC3b accumulation in absence of AP-2 does not appear to result from increased LC3b synthesis. A similar increase in folimycin-treated AP-2 KO neurons was observed for Rab7-positive puncta (Fig. 3n). These results argue that the accumulation of late-stage autophagosomes in AP-2 KO neurons is caused by decreased degradation of LC3b/Rab7-positive autophagosomes due to their defective retrograde transport.


Retrograde transport of TrkB-containing autophagosomes via the adaptor AP-2 mediates neuronal complexity and prevents neurodegeneration
AP-2 regulates autophagosome transport in neurons.(a) Time-lapse images of mRFP-LC3-positive puncta (arrows) in WT and AP-2μ KO neurons. Scale bar, 5 μm. (b) Kymographs of mRFP-LC3 carriers generated from a. (c) Average retrograde velocity of mRFP-LC3 carriers in WT and AP-2μ KO neurons. Loss of AP-2μ significantly decreased the LC3 velocity compared to WT controls (WT: 0.44±0.07 μm s−1, KO: 0.21±0.03 μm s−1, *P=0.019, n=4 independent experiments, 49–67 neurites per condition). (d–e) Electron micrographs of synapses from cultured WT and AP-2μ KO neurons. AP-2 KO neurons accumulate dense vesicular bodies with the majority representing concentric multilamellar structures (black boxes in d represent magnified areas in e). Scale bars, (d) 500 nm, (e) 100 nm. Sp, spine. See also Supplementary Fig. 3l–o. (f) Percentage of WT and KO synapses containing dense vesicular bodies (WT: 6.00%±1.54%, AP-2 KO: 13.00%±2.52%, *P=0.045, n=4, 100 synapses per condition). (g–i) Representative confocal images of cultured WT and AP-2 KO neurons immunostained for LC3b and Rab7 (white boxes in g represent the magnified areas in h,i). Scale bars, (g) 15 μm, (h,i) 2 μm. (j,k) Accumulation of LC3b-containing structures (LC3b puncta μm−2 are depicted, WT: 0.009±0.002, AP-2 KO: 0.040±0.009, *P=0.016, n=4, in total 39 AP-2 KO and 33 WT neurons) (j) and Rab7-containing structures (Rab7 puncta μm−2 are depicted, WT: 0.004±0.000, AP-2 KO: 0.007±0.000, *P=0.042, n=3, in total 29 AP-2 KO and 23 WT neurons) (k) in AP-2μ-KO neurons. (l) Enhanced colocalization of LC3b with Rab7 on neuronal autophagosomes in absence of AP-2μ based on Pearson's coefficient (Rp) (WT: 0.52±0.04, AP-2 KO: 0.64±0.01, *P=0.032). Rp was calculated for 64–84 regions of interest (ROI) per condition from three independent experiments (n=3). (m,n) Bar diagrams indicating similar numbers of LC3b- (WT: 0.09±0.01, AP-2 KO: 0.1±0.02) (m) and Rab7-positive puncta μm−2 (WT: 0.05±0.02%, AP-2 KO: 0.04±0.01) (n) in WT and AP-2μ KO neurons treated with folimycin.. Shown is the number of puncta per μm2 (n=3 independent experiments, 26 neurons per condition). Data in c,f,j–n are illustrated as box plots as described in Methods. Data reported in the text are mean±s.e.m. NS, non-significant.
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f3: AP-2 regulates autophagosome transport in neurons.(a) Time-lapse images of mRFP-LC3-positive puncta (arrows) in WT and AP-2μ KO neurons. Scale bar, 5 μm. (b) Kymographs of mRFP-LC3 carriers generated from a. (c) Average retrograde velocity of mRFP-LC3 carriers in WT and AP-2μ KO neurons. Loss of AP-2μ significantly decreased the LC3 velocity compared to WT controls (WT: 0.44±0.07 μm s−1, KO: 0.21±0.03 μm s−1, *P=0.019, n=4 independent experiments, 49–67 neurites per condition). (d–e) Electron micrographs of synapses from cultured WT and AP-2μ KO neurons. AP-2 KO neurons accumulate dense vesicular bodies with the majority representing concentric multilamellar structures (black boxes in d represent magnified areas in e). Scale bars, (d) 500 nm, (e) 100 nm. Sp, spine. See also Supplementary Fig. 3l–o. (f) Percentage of WT and KO synapses containing dense vesicular bodies (WT: 6.00%±1.54%, AP-2 KO: 13.00%±2.52%, *P=0.045, n=4, 100 synapses per condition). (g–i) Representative confocal images of cultured WT and AP-2 KO neurons immunostained for LC3b and Rab7 (white boxes in g represent the magnified areas in h,i). Scale bars, (g) 15 μm, (h,i) 2 μm. (j,k) Accumulation of LC3b-containing structures (LC3b puncta μm−2 are depicted, WT: 0.009±0.002, AP-2 KO: 0.040±0.009, *P=0.016, n=4, in total 39 AP-2 KO and 33 WT neurons) (j) and Rab7-containing structures (Rab7 puncta μm−2 are depicted, WT: 0.004±0.000, AP-2 KO: 0.007±0.000, *P=0.042, n=3, in total 29 AP-2 KO and 23 WT neurons) (k) in AP-2μ-KO neurons. (l) Enhanced colocalization of LC3b with Rab7 on neuronal autophagosomes in absence of AP-2μ based on Pearson's coefficient (Rp) (WT: 0.52±0.04, AP-2 KO: 0.64±0.01, *P=0.032). Rp was calculated for 64–84 regions of interest (ROI) per condition from three independent experiments (n=3). (m,n) Bar diagrams indicating similar numbers of LC3b- (WT: 0.09±0.01, AP-2 KO: 0.1±0.02) (m) and Rab7-positive puncta μm−2 (WT: 0.05±0.02%, AP-2 KO: 0.04±0.01) (n) in WT and AP-2μ KO neurons treated with folimycin.. Shown is the number of puncta per μm2 (n=3 independent experiments, 26 neurons per condition). Data in c,f,j–n are illustrated as box plots as described in Methods. Data reported in the text are mean±s.e.m. NS, non-significant.
Mentions: We then monitored the transport of autophagosomes in wild-type (WT) and AP-2μ KO neurons expressing mRFP-eGFP-LC3 by live imaging. In WT neurons mRFP-labelled autophagosomes displayed bidirectional movements with an average retrograde velocity of about 0.4–0.5 μm s−1 (Fig. 3a–c), similar to the values obtained for AP-2μ-mRFP with which it colocalizes (compare Fig. 1) and consistent with earlier data1032. AP-2μ deletion greatly reduced retrograde autophagosome velocity and the mobile fraction of retrograde LC3b-positive carriers (Fig. 3a–c, Supplementary Fig. 3e), while the fraction of stationary LC3b puncta was increased (Supplementary Fig. 3f). In agreement with the function of dynein motors in slow anterograde movement, lack of neuronal AP-2 caused a mild, yet statistically insignificant reduction in anterograde autophagosome transport (Supplementary Fig. 3e,g)33. Transport of mitochondria proceeded unperturbed in absence of neuronal AP-2 (Supplementary Fig. 3h–k). These data suggest that AP-2 regulates retrograde transport of autophagosomes from neurites to the cell soma, where most lysosomes are located510. Consistent with this hypothesis and with our live imaging results analysis of AP-2μ KO neurons by thin-section electron microcopy revealed an accumulation of dense vesicular and concentric multilamellar organelles resembling late-stage autophagosomes (also termed amphisomes) post-fusion with late endosomes (Fig. 3d–f, Supplementary Fig. 3o and below). As reported earlier26, AP-2μ KO neurons displayed a reduced number of synaptic vesicles (to about 60% of those seen in WT), in agreement with its canonical function in synaptic vesicle reformation. The ‘spheroid-like' accumulation of late-stage autophagosomes in neurites and in the soma of AP-2μ KO compared to WT control neurons was confirmed by immunostaining with antibodies against endogenous LC3b and late endosomal Rab7 (Fig. 3g–l for quantifications), indicating that the autophagosomal structures observed by light and electron microscopy have undergone fusion with late endosomes. In contrast, no significant alterations in the number or localization of early endosomes marked by Rab5 (Supplementary Fig. 4a,b) or LAMP1-positive late endosomes/lysosomes (Supplementary Fig. 3l–n, Supplementary Fig. 4c,d) were observed. To probe whether loss of AP-2 may alter LC3b synthesis and/or degradation3435, we treated WT and KO neurons with the lysosomotropic agent folimycin. Folimycin application caused the accumulation of LC3b-positive puncta in WT neurons to reach levels similar to those seen in neurons lacking AP-2μ (Fig. 3m, Supplementary Fig. 4e). Thus, LC3b accumulation in absence of AP-2 does not appear to result from increased LC3b synthesis. A similar increase in folimycin-treated AP-2 KO neurons was observed for Rab7-positive puncta (Fig. 3n). These results argue that the accumulation of late-stage autophagosomes in AP-2 KO neurons is caused by decreased degradation of LC3b/Rab7-positive autophagosomes due to their defective retrograde transport.

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.


Related in: MedlinePlus