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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.


Related in: MedlinePlus

AP-2 regulates autophagosome turnover independent of its role in endocytosis.(a) Tandem mRFP-eGFP-tagged LC3 as a reporter of autolysosome formation. (b) Mean mRFP/eGFP intensity ratio in control or serum-deprived WT or AP-2μ KO neurons (n=6 independent experiments, with 37–54 neurons per condition). No significant difference between WT and AP-2μ KO neurons was observed at steady state (P=0.398). Serum deprivation failed to trigger the formation of autolysosomes in AP-2μ neurons (control WT: 1.247±0.092, serum-deprived WT: 2.847±0.213, ***P<0.001, control KO: 1.528±0.130, serum-deprived KO: 1.774±0.169, P=0.640; serum-deprived WT versus serum-deprived KO **P=0.002). (c) Representative confocal images of WT and AP-2μ KO neurons immunostained for p62. Scale bars, 20 μm. (d) Increased number of p62-positive puncta per μm2 in AP-2μ KO (0.030±0.003) compared to WT neurons (0.017±0.003). *P=0.046, n=4, 33–39 neurons per condition. See also Supplementary Fig. 4g. (e) Average retrograde velocity of mRFP-LC3 carriers in control neurons expressing AP-2αA WT or LC3 binding-deficient AP-2αA Mut and co-expressing mRFP-eGFP-LC3. Loss of LC3-AP-2α binding significantly decreased LC3 transport compared to AP-2αA WT expressing controls (AP-2αAWT: 0.42±0.00 μm s−1, AP-2αAMut: 0.32±0.01 μm s−1, **P=0.007, n=3 independent experiments, ≥45–47 neurites per condition). (f) Mean mRFP/eGFP intensity ratio in control or serum-deprived neurons expressing AP-2αAWT or AP-2αAMut (n=4 independent experiments, ≥25 neurons per condition). No significant difference between neurons expressing AP-2αAWT or AP-2αAMut was observed at steady state (P=0.661). Serum deprivation fails to trigger autolysosome formation in neurons expressing AP-2αAMut (control AP-2αAMut: 1.544±0.361, serum-deprived AP-2αAMut: 1.775±0.088, P=0.886), but not in neurons expressing AP-2αAWT (control AP-2αAWT: 1.173±0.091, serum-deprived AP-2αAWT: 2.660±0.240, **P=0.003). (g,h) LC3-binding defective AP-2αA (AP-2αA Mut) restores clathrin-mediated endocytosis of transferrin in HeLa cells depleted of endogenous AP-2α (KD) (*P=0.041, **P=0.007, n=4, 156, 124, 148 cells per condition, respectively). Mean grey values of transferrin uptake in KD+AP-2αAWT and KD+AP-2αAMut conditions were normalized to KD condition set to 100%. Scale bar, 20 μm. Tf, transferrin. Data in b,d,e,f are illustrated as box plots as described in Methods. Data in h and all data reported in the text are mean±s.e.m. NS, non-significant.
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f4: AP-2 regulates autophagosome turnover independent of its role in endocytosis.(a) Tandem mRFP-eGFP-tagged LC3 as a reporter of autolysosome formation. (b) Mean mRFP/eGFP intensity ratio in control or serum-deprived WT or AP-2μ KO neurons (n=6 independent experiments, with 37–54 neurons per condition). No significant difference between WT and AP-2μ KO neurons was observed at steady state (P=0.398). Serum deprivation failed to trigger the formation of autolysosomes in AP-2μ neurons (control WT: 1.247±0.092, serum-deprived WT: 2.847±0.213, ***P<0.001, control KO: 1.528±0.130, serum-deprived KO: 1.774±0.169, P=0.640; serum-deprived WT versus serum-deprived KO **P=0.002). (c) Representative confocal images of WT and AP-2μ KO neurons immunostained for p62. Scale bars, 20 μm. (d) Increased number of p62-positive puncta per μm2 in AP-2μ KO (0.030±0.003) compared to WT neurons (0.017±0.003). *P=0.046, n=4, 33–39 neurons per condition. See also Supplementary Fig. 4g. (e) Average retrograde velocity of mRFP-LC3 carriers in control neurons expressing AP-2αA WT or LC3 binding-deficient AP-2αA Mut and co-expressing mRFP-eGFP-LC3. Loss of LC3-AP-2α binding significantly decreased LC3 transport compared to AP-2αA WT expressing controls (AP-2αAWT: 0.42±0.00 μm s−1, AP-2αAMut: 0.32±0.01 μm s−1, **P=0.007, n=3 independent experiments, ≥45–47 neurites per condition). (f) Mean mRFP/eGFP intensity ratio in control or serum-deprived neurons expressing AP-2αAWT or AP-2αAMut (n=4 independent experiments, ≥25 neurons per condition). No significant difference between neurons expressing AP-2αAWT or AP-2αAMut was observed at steady state (P=0.661). Serum deprivation fails to trigger autolysosome formation in neurons expressing AP-2αAMut (control AP-2αAMut: 1.544±0.361, serum-deprived AP-2αAMut: 1.775±0.088, P=0.886), but not in neurons expressing AP-2αAWT (control AP-2αAWT: 1.173±0.091, serum-deprived AP-2αAWT: 2.660±0.240, **P=0.003). (g,h) LC3-binding defective AP-2αA (AP-2αA Mut) restores clathrin-mediated endocytosis of transferrin in HeLa cells depleted of endogenous AP-2α (KD) (*P=0.041, **P=0.007, n=4, 156, 124, 148 cells per condition, respectively). Mean grey values of transferrin uptake in KD+AP-2αAWT and KD+AP-2αAMut conditions were normalized to KD condition set to 100%. Scale bar, 20 μm. Tf, transferrin. Data in b,d,e,f are illustrated as box plots as described in Methods. Data in h and all data reported in the text are mean±s.e.m. NS, non-significant.

Mentions: As decreased degradation of LC3b/Rab7-positive autophagosomes in the absence of AP-2μ might result from their defective delivery to lysosomes, we next probed the turnover of autophagosomes in the absence of AP-2μ using mRFP-eGFP-LC3 as a reporter (Fig. 4a). Serum deprivation caused an elevation of the mRFP/eGFP fluorescent ratio in WT neurons indicative of increased starvation-induced autophagic flux. In contrast, no significant changes in the mRFP/eGFP ratio were observed in AP-2μ KO neurons (Fig. 4b, Supplementary Fig. 4f). Moreover, AP-2 loss resulted in the accumulation of the autophagic adaptor and degradative substrate p62/SQSTM1 (Fig. 4c,d) that persisted upon inhibition of protein synthesis in the presence of cylcoheximide (Supplementary Fig. 4g), suggesting that it is the result of defective autophagosome turnover. Consistent with this, mTORC1 signalling measured by phospho-S6 kinase 1 and phospho-Raptor levels was not significantly changed in AP-2 KO brains, although we detected a slight increase in the total amount of S6 kinase (Supplementary Fig. 4h,i). These data suggest that autophagosome accumulation in absence of AP-2 is not a consequence of reduced mTORC1 activity, a key repressor of autophagosome formation.


Retrograde transport of TrkB-containing autophagosomes via the adaptor AP-2 mediates neuronal complexity and prevents neurodegeneration
AP-2 regulates autophagosome turnover independent of its role in endocytosis.(a) Tandem mRFP-eGFP-tagged LC3 as a reporter of autolysosome formation. (b) Mean mRFP/eGFP intensity ratio in control or serum-deprived WT or AP-2μ KO neurons (n=6 independent experiments, with 37–54 neurons per condition). No significant difference between WT and AP-2μ KO neurons was observed at steady state (P=0.398). Serum deprivation failed to trigger the formation of autolysosomes in AP-2μ neurons (control WT: 1.247±0.092, serum-deprived WT: 2.847±0.213, ***P<0.001, control KO: 1.528±0.130, serum-deprived KO: 1.774±0.169, P=0.640; serum-deprived WT versus serum-deprived KO **P=0.002). (c) Representative confocal images of WT and AP-2μ KO neurons immunostained for p62. Scale bars, 20 μm. (d) Increased number of p62-positive puncta per μm2 in AP-2μ KO (0.030±0.003) compared to WT neurons (0.017±0.003). *P=0.046, n=4, 33–39 neurons per condition. See also Supplementary Fig. 4g. (e) Average retrograde velocity of mRFP-LC3 carriers in control neurons expressing AP-2αA WT or LC3 binding-deficient AP-2αA Mut and co-expressing mRFP-eGFP-LC3. Loss of LC3-AP-2α binding significantly decreased LC3 transport compared to AP-2αA WT expressing controls (AP-2αAWT: 0.42±0.00 μm s−1, AP-2αAMut: 0.32±0.01 μm s−1, **P=0.007, n=3 independent experiments, ≥45–47 neurites per condition). (f) Mean mRFP/eGFP intensity ratio in control or serum-deprived neurons expressing AP-2αAWT or AP-2αAMut (n=4 independent experiments, ≥25 neurons per condition). No significant difference between neurons expressing AP-2αAWT or AP-2αAMut was observed at steady state (P=0.661). Serum deprivation fails to trigger autolysosome formation in neurons expressing AP-2αAMut (control AP-2αAMut: 1.544±0.361, serum-deprived AP-2αAMut: 1.775±0.088, P=0.886), but not in neurons expressing AP-2αAWT (control AP-2αAWT: 1.173±0.091, serum-deprived AP-2αAWT: 2.660±0.240, **P=0.003). (g,h) LC3-binding defective AP-2αA (AP-2αA Mut) restores clathrin-mediated endocytosis of transferrin in HeLa cells depleted of endogenous AP-2α (KD) (*P=0.041, **P=0.007, n=4, 156, 124, 148 cells per condition, respectively). Mean grey values of transferrin uptake in KD+AP-2αAWT and KD+AP-2αAMut conditions were normalized to KD condition set to 100%. Scale bar, 20 μm. Tf, transferrin. Data in b,d,e,f are illustrated as box plots as described in Methods. Data in h and all data reported in the text are mean±s.e.m. NS, non-significant.
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f4: AP-2 regulates autophagosome turnover independent of its role in endocytosis.(a) Tandem mRFP-eGFP-tagged LC3 as a reporter of autolysosome formation. (b) Mean mRFP/eGFP intensity ratio in control or serum-deprived WT or AP-2μ KO neurons (n=6 independent experiments, with 37–54 neurons per condition). No significant difference between WT and AP-2μ KO neurons was observed at steady state (P=0.398). Serum deprivation failed to trigger the formation of autolysosomes in AP-2μ neurons (control WT: 1.247±0.092, serum-deprived WT: 2.847±0.213, ***P<0.001, control KO: 1.528±0.130, serum-deprived KO: 1.774±0.169, P=0.640; serum-deprived WT versus serum-deprived KO **P=0.002). (c) Representative confocal images of WT and AP-2μ KO neurons immunostained for p62. Scale bars, 20 μm. (d) Increased number of p62-positive puncta per μm2 in AP-2μ KO (0.030±0.003) compared to WT neurons (0.017±0.003). *P=0.046, n=4, 33–39 neurons per condition. See also Supplementary Fig. 4g. (e) Average retrograde velocity of mRFP-LC3 carriers in control neurons expressing AP-2αA WT or LC3 binding-deficient AP-2αA Mut and co-expressing mRFP-eGFP-LC3. Loss of LC3-AP-2α binding significantly decreased LC3 transport compared to AP-2αA WT expressing controls (AP-2αAWT: 0.42±0.00 μm s−1, AP-2αAMut: 0.32±0.01 μm s−1, **P=0.007, n=3 independent experiments, ≥45–47 neurites per condition). (f) Mean mRFP/eGFP intensity ratio in control or serum-deprived neurons expressing AP-2αAWT or AP-2αAMut (n=4 independent experiments, ≥25 neurons per condition). No significant difference between neurons expressing AP-2αAWT or AP-2αAMut was observed at steady state (P=0.661). Serum deprivation fails to trigger autolysosome formation in neurons expressing AP-2αAMut (control AP-2αAMut: 1.544±0.361, serum-deprived AP-2αAMut: 1.775±0.088, P=0.886), but not in neurons expressing AP-2αAWT (control AP-2αAWT: 1.173±0.091, serum-deprived AP-2αAWT: 2.660±0.240, **P=0.003). (g,h) LC3-binding defective AP-2αA (AP-2αA Mut) restores clathrin-mediated endocytosis of transferrin in HeLa cells depleted of endogenous AP-2α (KD) (*P=0.041, **P=0.007, n=4, 156, 124, 148 cells per condition, respectively). Mean grey values of transferrin uptake in KD+AP-2αAWT and KD+AP-2αAMut conditions were normalized to KD condition set to 100%. Scale bar, 20 μm. Tf, transferrin. Data in b,d,e,f are illustrated as box plots as described in Methods. Data in h and all data reported in the text are mean±s.e.m. NS, non-significant.
Mentions: As decreased degradation of LC3b/Rab7-positive autophagosomes in the absence of AP-2μ might result from their defective delivery to lysosomes, we next probed the turnover of autophagosomes in the absence of AP-2μ using mRFP-eGFP-LC3 as a reporter (Fig. 4a). Serum deprivation caused an elevation of the mRFP/eGFP fluorescent ratio in WT neurons indicative of increased starvation-induced autophagic flux. In contrast, no significant changes in the mRFP/eGFP ratio were observed in AP-2μ KO neurons (Fig. 4b, Supplementary Fig. 4f). Moreover, AP-2 loss resulted in the accumulation of the autophagic adaptor and degradative substrate p62/SQSTM1 (Fig. 4c,d) that persisted upon inhibition of protein synthesis in the presence of cylcoheximide (Supplementary Fig. 4g), suggesting that it is the result of defective autophagosome turnover. Consistent with this, mTORC1 signalling measured by phospho-S6 kinase 1 and phospho-Raptor levels was not significantly changed in AP-2 KO brains, although we detected a slight increase in the total amount of S6 kinase (Supplementary Fig. 4h,i). These data suggest that autophagosome accumulation in absence of AP-2 is not a consequence of reduced mTORC1 activity, a key repressor of autophagosome formation.

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