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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

Reduced neuronal complexity and neurodegeneration in the absence of neuronal AP-2μ in vivo.(a) Postnatal growth retardation of 21-day-old KO mice conditionally deleted for AP-2μ by transgenic expression of Cre recombinase under the neuron-specific Tα1 tubulin promoter (AP-2lox/lox:Tubα1-Cre ). See also Supplementary Figs 7a,b. (b) Kaplan–Meier survival curves of neuron-specific AP-2μ KO mice and littermate controls (AP-2wt/wt:Tubα1-Cre (WT), AP-2lox/wt:Tubα1-Cre (Het) and AP-2lox/lox:Tubα1-Cre (KO)). (c,d) Golgi silver impregnation of cortices from p20 WT and AP-2μ KO mice reveal the loss of dendritic architecture in AP-2μ KO brain. Scale bars, 200 μm. (e,f) 3D morphology of stellate neurons in control (e) and AP-2μ-deficient (f) brains. Scale bars, 40 μm. (g,h) Sholl analysis of stellate cells, revealing their branching complexity (g) and total dendritic length (h) in p20 WT and AP-2μ KO brains. (i) Histopathological analysis of the brain of AP-2μ KO mice at p20 shows marked degeneration of the thalamus (indicated by dotted line), but no overt alteration of the hippocampus (CA1). Nissl-stained brain sections of WT and conditional AP-2μ KO mice. cc, corpus callosum; AV, anteroventral thalamic nucleus; AM, anteromedial thalamic nucleus; LD, laterodorsal thalamic nucleus; VL, ventrolateral thalamic nucleus; VM, ventromedial thalamic nucleus; VPL, ventral posteriorlateral thalamic nucleus, Re, reuniens thalamic nucleus; Rt, reticular nucleus. Scale bars, 800 μm. See also Supplementary Fig. 7g–p for an overview of the temporal progression of neurodegeneration in the brain of AP-2 KO mice. (j) Loss of barrel compartments in the somatosensory cortex of AP-2μ KO mice. Cortical barrels visualized by immunostaining for potassium-chloride transporter member 2 are seen in WT controls, but absent in AP-2μ KO brains. Roman numbers indicate cortical layers. Scale bars, 500 μm. (k,l) Representative confocal images of thalamus in WT and AP-2μ KO mice immunostained for p62. White rectangular boxes in k indicate areas magnified in l. Scale bars, 5 μm. (m) Mean size of p62-positive puncta is significantly increased in brains of AP-2μ KO mice (1.92±0.26) compared to WT littermates (1.26±0.08, *P=0.041, n=5). Data in m are illustrated as box plots as described in Methods. Data in b,g,h and all data reported in the text are mean±s.e.m.
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f6: Reduced neuronal complexity and neurodegeneration in the absence of neuronal AP-2μ in vivo.(a) Postnatal growth retardation of 21-day-old KO mice conditionally deleted for AP-2μ by transgenic expression of Cre recombinase under the neuron-specific Tα1 tubulin promoter (AP-2lox/lox:Tubα1-Cre ). See also Supplementary Figs 7a,b. (b) Kaplan–Meier survival curves of neuron-specific AP-2μ KO mice and littermate controls (AP-2wt/wt:Tubα1-Cre (WT), AP-2lox/wt:Tubα1-Cre (Het) and AP-2lox/lox:Tubα1-Cre (KO)). (c,d) Golgi silver impregnation of cortices from p20 WT and AP-2μ KO mice reveal the loss of dendritic architecture in AP-2μ KO brain. Scale bars, 200 μm. (e,f) 3D morphology of stellate neurons in control (e) and AP-2μ-deficient (f) brains. Scale bars, 40 μm. (g,h) Sholl analysis of stellate cells, revealing their branching complexity (g) and total dendritic length (h) in p20 WT and AP-2μ KO brains. (i) Histopathological analysis of the brain of AP-2μ KO mice at p20 shows marked degeneration of the thalamus (indicated by dotted line), but no overt alteration of the hippocampus (CA1). Nissl-stained brain sections of WT and conditional AP-2μ KO mice. cc, corpus callosum; AV, anteroventral thalamic nucleus; AM, anteromedial thalamic nucleus; LD, laterodorsal thalamic nucleus; VL, ventrolateral thalamic nucleus; VM, ventromedial thalamic nucleus; VPL, ventral posteriorlateral thalamic nucleus, Re, reuniens thalamic nucleus; Rt, reticular nucleus. Scale bars, 800 μm. See also Supplementary Fig. 7g–p for an overview of the temporal progression of neurodegeneration in the brain of AP-2 KO mice. (j) Loss of barrel compartments in the somatosensory cortex of AP-2μ KO mice. Cortical barrels visualized by immunostaining for potassium-chloride transporter member 2 are seen in WT controls, but absent in AP-2μ KO brains. Roman numbers indicate cortical layers. Scale bars, 500 μm. (k,l) Representative confocal images of thalamus in WT and AP-2μ KO mice immunostained for p62. White rectangular boxes in k indicate areas magnified in l. Scale bars, 5 μm. (m) Mean size of p62-positive puncta is significantly increased in brains of AP-2μ KO mice (1.92±0.26) compared to WT littermates (1.26±0.08, *P=0.041, n=5). Data in m are illustrated as box plots as described in Methods. Data in b,g,h and all data reported in the text are mean±s.e.m.

Mentions: Given the prominent defects in transport of TrkB-containing autophagosomes and neuronal arborization observed in hippocampal neurons in culture following acute inactivation of AP-2μ, we wanted to explore the functional importance of AP-2 for TrkB-mediated neuronal arborization in the brain in vivo. To specifically ablate AP-2μ expression in CNS neurons we crossed floxed AP-2μ mice with a strain expressing Cre under the neuron-specific tubulin α1 promoter41 starting from embryonic day 13.5 (AP-2μlox/lox:Tubα1-Cre mice). Conditional AP-2μ KO mice were born well below Mendelian ratios (KO: 16% instead of 25% as expected, P<0.0011, see Supplementary Fig. 7a) and lagged behind in postnatal development including cessation of weight gain at about 2 weeks and postnatal lethality between postnatal day (p) 21 and 26 (Fig. 6a,b, Supplementary Fig. 7b). To analyse the role of AP-2 in dendritic arborization in vivo we visualized the branching complexity of neurons in the cortex of p20 control and AP-2 KO mice by Golgi silver impregnation. Neuron-specific AP-2μ KO mice displayed dramatic defects in dendritic architecture compared to WT littermates (Fig. 6c–f), as evident from Sholl analysis of stellate cells in cortical layers III and II, respectively (Fig. 6g,h). These data confirm our observations from cultured AP-2μ KO neurons and suggest that neuronal AP-2 is required for neuronal arborization in vivo.


Retrograde transport of TrkB-containing autophagosomes via the adaptor AP-2 mediates neuronal complexity and prevents neurodegeneration
Reduced neuronal complexity and neurodegeneration in the absence of neuronal AP-2μ in vivo.(a) Postnatal growth retardation of 21-day-old KO mice conditionally deleted for AP-2μ by transgenic expression of Cre recombinase under the neuron-specific Tα1 tubulin promoter (AP-2lox/lox:Tubα1-Cre ). See also Supplementary Figs 7a,b. (b) Kaplan–Meier survival curves of neuron-specific AP-2μ KO mice and littermate controls (AP-2wt/wt:Tubα1-Cre (WT), AP-2lox/wt:Tubα1-Cre (Het) and AP-2lox/lox:Tubα1-Cre (KO)). (c,d) Golgi silver impregnation of cortices from p20 WT and AP-2μ KO mice reveal the loss of dendritic architecture in AP-2μ KO brain. Scale bars, 200 μm. (e,f) 3D morphology of stellate neurons in control (e) and AP-2μ-deficient (f) brains. Scale bars, 40 μm. (g,h) Sholl analysis of stellate cells, revealing their branching complexity (g) and total dendritic length (h) in p20 WT and AP-2μ KO brains. (i) Histopathological analysis of the brain of AP-2μ KO mice at p20 shows marked degeneration of the thalamus (indicated by dotted line), but no overt alteration of the hippocampus (CA1). Nissl-stained brain sections of WT and conditional AP-2μ KO mice. cc, corpus callosum; AV, anteroventral thalamic nucleus; AM, anteromedial thalamic nucleus; LD, laterodorsal thalamic nucleus; VL, ventrolateral thalamic nucleus; VM, ventromedial thalamic nucleus; VPL, ventral posteriorlateral thalamic nucleus, Re, reuniens thalamic nucleus; Rt, reticular nucleus. Scale bars, 800 μm. See also Supplementary Fig. 7g–p for an overview of the temporal progression of neurodegeneration in the brain of AP-2 KO mice. (j) Loss of barrel compartments in the somatosensory cortex of AP-2μ KO mice. Cortical barrels visualized by immunostaining for potassium-chloride transporter member 2 are seen in WT controls, but absent in AP-2μ KO brains. Roman numbers indicate cortical layers. Scale bars, 500 μm. (k,l) Representative confocal images of thalamus in WT and AP-2μ KO mice immunostained for p62. White rectangular boxes in k indicate areas magnified in l. Scale bars, 5 μm. (m) Mean size of p62-positive puncta is significantly increased in brains of AP-2μ KO mice (1.92±0.26) compared to WT littermates (1.26±0.08, *P=0.041, n=5). Data in m are illustrated as box plots as described in Methods. Data in b,g,h and all data reported in the text are mean±s.e.m.
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f6: Reduced neuronal complexity and neurodegeneration in the absence of neuronal AP-2μ in vivo.(a) Postnatal growth retardation of 21-day-old KO mice conditionally deleted for AP-2μ by transgenic expression of Cre recombinase under the neuron-specific Tα1 tubulin promoter (AP-2lox/lox:Tubα1-Cre ). See also Supplementary Figs 7a,b. (b) Kaplan–Meier survival curves of neuron-specific AP-2μ KO mice and littermate controls (AP-2wt/wt:Tubα1-Cre (WT), AP-2lox/wt:Tubα1-Cre (Het) and AP-2lox/lox:Tubα1-Cre (KO)). (c,d) Golgi silver impregnation of cortices from p20 WT and AP-2μ KO mice reveal the loss of dendritic architecture in AP-2μ KO brain. Scale bars, 200 μm. (e,f) 3D morphology of stellate neurons in control (e) and AP-2μ-deficient (f) brains. Scale bars, 40 μm. (g,h) Sholl analysis of stellate cells, revealing their branching complexity (g) and total dendritic length (h) in p20 WT and AP-2μ KO brains. (i) Histopathological analysis of the brain of AP-2μ KO mice at p20 shows marked degeneration of the thalamus (indicated by dotted line), but no overt alteration of the hippocampus (CA1). Nissl-stained brain sections of WT and conditional AP-2μ KO mice. cc, corpus callosum; AV, anteroventral thalamic nucleus; AM, anteromedial thalamic nucleus; LD, laterodorsal thalamic nucleus; VL, ventrolateral thalamic nucleus; VM, ventromedial thalamic nucleus; VPL, ventral posteriorlateral thalamic nucleus, Re, reuniens thalamic nucleus; Rt, reticular nucleus. Scale bars, 800 μm. See also Supplementary Fig. 7g–p for an overview of the temporal progression of neurodegeneration in the brain of AP-2 KO mice. (j) Loss of barrel compartments in the somatosensory cortex of AP-2μ KO mice. Cortical barrels visualized by immunostaining for potassium-chloride transporter member 2 are seen in WT controls, but absent in AP-2μ KO brains. Roman numbers indicate cortical layers. Scale bars, 500 μm. (k,l) Representative confocal images of thalamus in WT and AP-2μ KO mice immunostained for p62. White rectangular boxes in k indicate areas magnified in l. Scale bars, 5 μm. (m) Mean size of p62-positive puncta is significantly increased in brains of AP-2μ KO mice (1.92±0.26) compared to WT littermates (1.26±0.08, *P=0.041, n=5). Data in m are illustrated as box plots as described in Methods. Data in b,g,h and all data reported in the text are mean±s.e.m.
Mentions: Given the prominent defects in transport of TrkB-containing autophagosomes and neuronal arborization observed in hippocampal neurons in culture following acute inactivation of AP-2μ, we wanted to explore the functional importance of AP-2 for TrkB-mediated neuronal arborization in the brain in vivo. To specifically ablate AP-2μ expression in CNS neurons we crossed floxed AP-2μ mice with a strain expressing Cre under the neuron-specific tubulin α1 promoter41 starting from embryonic day 13.5 (AP-2μlox/lox:Tubα1-Cre mice). Conditional AP-2μ KO mice were born well below Mendelian ratios (KO: 16% instead of 25% as expected, P<0.0011, see Supplementary Fig. 7a) and lagged behind in postnatal development including cessation of weight gain at about 2 weeks and postnatal lethality between postnatal day (p) 21 and 26 (Fig. 6a,b, Supplementary Fig. 7b). To analyse the role of AP-2 in dendritic arborization in vivo we visualized the branching complexity of neurons in the cortex of p20 control and AP-2 KO mice by Golgi silver impregnation. Neuron-specific AP-2μ KO mice displayed dramatic defects in dendritic architecture compared to WT littermates (Fig. 6c–f), as evident from Sholl analysis of stellate cells in cortical layers III and II, respectively (Fig. 6g,h). These data confirm our observations from cultured AP-2μ KO neurons and suggest that neuronal AP-2 is required for neuronal arborization in vivo.

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