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Uptake and recycling of pro-BDNF for transmitter-induced secretion by cortical astrocytes.

Bergami M, Santi S, Formaggio E, Cagnoli C, Verderio C, Blum R, Berninger B, Matteoli M, Canossa M - J. Cell Biol. (2008)

Bottom Line: Fluorescence-tagged pro-BDNF and real-time total internal reflection fluorescence microscopy in cultured astrocytes is used to monitor single endocytic vesicles in response to the neurotransmitter glutamate.We find that endocytosed pro-BDNF is routed into a fast recycling pathway for subsequent soluble NSF attachment protein receptor-dependent secretion.Thus, astrocytes contain an endocytic compartment competent for pro-BDNF recycling, suggesting a specialized form of bidirectional communication between neurons and glia.

View Article: PubMed Central - PubMed

Affiliation: Department of Human and General Physiology, University of Bologna, I-40126 Bologna, Italy.

ABSTRACT
Activity-dependent secretion of brain-derived neurotrophic factor (BDNF) is thought to enhance synaptic plasticity, but the mechanisms controlling extracellular availability and clearance of secreted BDNF are poorly understood. We show that BDNF is secreted in its precursor form (pro-BDNF) and is then cleared from the extracellular space through rapid uptake by nearby astrocytes after theta-burst stimulation in layer II/III of cortical slices, a paradigm resulting in long-term potentiation of synaptic transmission. Internalization of pro-BDNF occurs via the formation of a complex with the pan-neurotrophin receptor p75 and subsequent clathrin-dependent endocytosis. Fluorescence-tagged pro-BDNF and real-time total internal reflection fluorescence microscopy in cultured astrocytes is used to monitor single endocytic vesicles in response to the neurotransmitter glutamate. We find that endocytosed pro-BDNF is routed into a fast recycling pathway for subsequent soluble NSF attachment protein receptor-dependent secretion. Thus, astrocytes contain an endocytic compartment competent for pro-BDNF recycling, suggesting a specialized form of bidirectional communication between neurons and glia.

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Transfer of pro-BDNF from neurons to perineuronal astrocytes. (A) Schematic representation of the slice preparation. (B) Western blot analysis of BDNF (mix) or cleavage-resistant pro-BDNF (Mowla et al., 2001) using α-BDNF– or α–pro-BDNF–specific antibodies. (C) Field potential amplitudes (black circles) and BDNF levels (gray circles) upon basal (control) or TBS stimulations. After recording, slices were immunostained using α–pro-BDNF. Immunoreactivity is shown in two adjacent areas corresponding to areas A1 and A2 of A. (D) Immunohistochemistry using α-BDNF. Bars, 100 μm. (E) High resolution confocal images of A1 in a slice 20 min after TBS. Pro-BDNF immunoreactivity is shown at the site of astrocytic contact with a neuron (box and inset 1), the astrocytic cell body (box and inset 2), and processes (box and inset 3). Colocalization of pro-BDNF with GFAP immunoreactivity is shown and superimposed onto the 3D reconstruction of the GFAP signal. Arrowheads indicate pro-BDNF immunoreactive puncta distributed along the astrocytic processes. Bar, 20 μm. (F) Time course of pro-BDNF/GFAP colocalization (four slices and nine cells). (G) Pro-BDNF/GFAP colocalization in astrocytes of control (three slices and 12 cells) or TBS slices in the absence (six slices and 24 cells) or presence of anisomycin (five slices and 11 cells), TrkB-Fc (four slices and nine cells), and plasmin (five slices and 24 cells) 10 min after stimulation. NeuN, neuronal nuclei. Data are means ± SEM (error bars). *, P ≤ 0.05.
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fig1: Transfer of pro-BDNF from neurons to perineuronal astrocytes. (A) Schematic representation of the slice preparation. (B) Western blot analysis of BDNF (mix) or cleavage-resistant pro-BDNF (Mowla et al., 2001) using α-BDNF– or α–pro-BDNF–specific antibodies. (C) Field potential amplitudes (black circles) and BDNF levels (gray circles) upon basal (control) or TBS stimulations. After recording, slices were immunostained using α–pro-BDNF. Immunoreactivity is shown in two adjacent areas corresponding to areas A1 and A2 of A. (D) Immunohistochemistry using α-BDNF. Bars, 100 μm. (E) High resolution confocal images of A1 in a slice 20 min after TBS. Pro-BDNF immunoreactivity is shown at the site of astrocytic contact with a neuron (box and inset 1), the astrocytic cell body (box and inset 2), and processes (box and inset 3). Colocalization of pro-BDNF with GFAP immunoreactivity is shown and superimposed onto the 3D reconstruction of the GFAP signal. Arrowheads indicate pro-BDNF immunoreactive puncta distributed along the astrocytic processes. Bar, 20 μm. (F) Time course of pro-BDNF/GFAP colocalization (four slices and nine cells). (G) Pro-BDNF/GFAP colocalization in astrocytes of control (three slices and 12 cells) or TBS slices in the absence (six slices and 24 cells) or presence of anisomycin (five slices and 11 cells), TrkB-Fc (four slices and nine cells), and plasmin (five slices and 24 cells) 10 min after stimulation. NeuN, neuronal nuclei. Data are means ± SEM (error bars). *, P ≤ 0.05.

Mentions: Field recordings were performed in layers II/III of rat perirhinal cortex slices (Fig. 1 A) subjected to either basal (0.033 Hz) or TBS (100 Hz) stimulation. BDNF secretion was measured in the collected perfusion medium by ELISA (Fig. 1 C). Although BDNF levels remained constant during basal stimulation, TBS induced a rapid, transient increase in BDNF in the perfusate, which correlated with induction of synaptic potentiation of the field potential as described previously (Aicardi et al., 2004). Fig. 1 C shows representative examples of pro-BDNF immunoreactivity in the perirhinal cortex upon basal and TBS stimulation using an antibody directed specifically (Fig. 1 B) against the pro-region of the neurotrophin. Basal levels of pro-BDNF were detected proximal to (Fig. 1 A, A1) and distal from (Fig. 1 A, A2) the stimulation electrode in controls. A marked increase in pro-BDNF immunoreactivity was observed after TBS, which gradually declined distally from the stimulation electrode. This effect was blocked by prior treatment with the protein synthesis inhibitor anisomycin (unpublished data), consistent with the pro-BDNF increase depending on activity-dependent local protein synthesis (Kandel, 2001). Similar results were obtained using an antibody directed against the mature portion of the neurotrophin that recognizes both mature and pro-BDNF (Fig. 1 D).


Uptake and recycling of pro-BDNF for transmitter-induced secretion by cortical astrocytes.

Bergami M, Santi S, Formaggio E, Cagnoli C, Verderio C, Blum R, Berninger B, Matteoli M, Canossa M - J. Cell Biol. (2008)

Transfer of pro-BDNF from neurons to perineuronal astrocytes. (A) Schematic representation of the slice preparation. (B) Western blot analysis of BDNF (mix) or cleavage-resistant pro-BDNF (Mowla et al., 2001) using α-BDNF– or α–pro-BDNF–specific antibodies. (C) Field potential amplitudes (black circles) and BDNF levels (gray circles) upon basal (control) or TBS stimulations. After recording, slices were immunostained using α–pro-BDNF. Immunoreactivity is shown in two adjacent areas corresponding to areas A1 and A2 of A. (D) Immunohistochemistry using α-BDNF. Bars, 100 μm. (E) High resolution confocal images of A1 in a slice 20 min after TBS. Pro-BDNF immunoreactivity is shown at the site of astrocytic contact with a neuron (box and inset 1), the astrocytic cell body (box and inset 2), and processes (box and inset 3). Colocalization of pro-BDNF with GFAP immunoreactivity is shown and superimposed onto the 3D reconstruction of the GFAP signal. Arrowheads indicate pro-BDNF immunoreactive puncta distributed along the astrocytic processes. Bar, 20 μm. (F) Time course of pro-BDNF/GFAP colocalization (four slices and nine cells). (G) Pro-BDNF/GFAP colocalization in astrocytes of control (three slices and 12 cells) or TBS slices in the absence (six slices and 24 cells) or presence of anisomycin (five slices and 11 cells), TrkB-Fc (four slices and nine cells), and plasmin (five slices and 24 cells) 10 min after stimulation. NeuN, neuronal nuclei. Data are means ± SEM (error bars). *, P ≤ 0.05.
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Related In: Results  -  Collection

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fig1: Transfer of pro-BDNF from neurons to perineuronal astrocytes. (A) Schematic representation of the slice preparation. (B) Western blot analysis of BDNF (mix) or cleavage-resistant pro-BDNF (Mowla et al., 2001) using α-BDNF– or α–pro-BDNF–specific antibodies. (C) Field potential amplitudes (black circles) and BDNF levels (gray circles) upon basal (control) or TBS stimulations. After recording, slices were immunostained using α–pro-BDNF. Immunoreactivity is shown in two adjacent areas corresponding to areas A1 and A2 of A. (D) Immunohistochemistry using α-BDNF. Bars, 100 μm. (E) High resolution confocal images of A1 in a slice 20 min after TBS. Pro-BDNF immunoreactivity is shown at the site of astrocytic contact with a neuron (box and inset 1), the astrocytic cell body (box and inset 2), and processes (box and inset 3). Colocalization of pro-BDNF with GFAP immunoreactivity is shown and superimposed onto the 3D reconstruction of the GFAP signal. Arrowheads indicate pro-BDNF immunoreactive puncta distributed along the astrocytic processes. Bar, 20 μm. (F) Time course of pro-BDNF/GFAP colocalization (four slices and nine cells). (G) Pro-BDNF/GFAP colocalization in astrocytes of control (three slices and 12 cells) or TBS slices in the absence (six slices and 24 cells) or presence of anisomycin (five slices and 11 cells), TrkB-Fc (four slices and nine cells), and plasmin (five slices and 24 cells) 10 min after stimulation. NeuN, neuronal nuclei. Data are means ± SEM (error bars). *, P ≤ 0.05.
Mentions: Field recordings were performed in layers II/III of rat perirhinal cortex slices (Fig. 1 A) subjected to either basal (0.033 Hz) or TBS (100 Hz) stimulation. BDNF secretion was measured in the collected perfusion medium by ELISA (Fig. 1 C). Although BDNF levels remained constant during basal stimulation, TBS induced a rapid, transient increase in BDNF in the perfusate, which correlated with induction of synaptic potentiation of the field potential as described previously (Aicardi et al., 2004). Fig. 1 C shows representative examples of pro-BDNF immunoreactivity in the perirhinal cortex upon basal and TBS stimulation using an antibody directed specifically (Fig. 1 B) against the pro-region of the neurotrophin. Basal levels of pro-BDNF were detected proximal to (Fig. 1 A, A1) and distal from (Fig. 1 A, A2) the stimulation electrode in controls. A marked increase in pro-BDNF immunoreactivity was observed after TBS, which gradually declined distally from the stimulation electrode. This effect was blocked by prior treatment with the protein synthesis inhibitor anisomycin (unpublished data), consistent with the pro-BDNF increase depending on activity-dependent local protein synthesis (Kandel, 2001). Similar results were obtained using an antibody directed against the mature portion of the neurotrophin that recognizes both mature and pro-BDNF (Fig. 1 D).

Bottom Line: Fluorescence-tagged pro-BDNF and real-time total internal reflection fluorescence microscopy in cultured astrocytes is used to monitor single endocytic vesicles in response to the neurotransmitter glutamate.We find that endocytosed pro-BDNF is routed into a fast recycling pathway for subsequent soluble NSF attachment protein receptor-dependent secretion.Thus, astrocytes contain an endocytic compartment competent for pro-BDNF recycling, suggesting a specialized form of bidirectional communication between neurons and glia.

View Article: PubMed Central - PubMed

Affiliation: Department of Human and General Physiology, University of Bologna, I-40126 Bologna, Italy.

ABSTRACT
Activity-dependent secretion of brain-derived neurotrophic factor (BDNF) is thought to enhance synaptic plasticity, but the mechanisms controlling extracellular availability and clearance of secreted BDNF are poorly understood. We show that BDNF is secreted in its precursor form (pro-BDNF) and is then cleared from the extracellular space through rapid uptake by nearby astrocytes after theta-burst stimulation in layer II/III of cortical slices, a paradigm resulting in long-term potentiation of synaptic transmission. Internalization of pro-BDNF occurs via the formation of a complex with the pan-neurotrophin receptor p75 and subsequent clathrin-dependent endocytosis. Fluorescence-tagged pro-BDNF and real-time total internal reflection fluorescence microscopy in cultured astrocytes is used to monitor single endocytic vesicles in response to the neurotransmitter glutamate. We find that endocytosed pro-BDNF is routed into a fast recycling pathway for subsequent soluble NSF attachment protein receptor-dependent secretion. Thus, astrocytes contain an endocytic compartment competent for pro-BDNF recycling, suggesting a specialized form of bidirectional communication between neurons and glia.

Show MeSH
Related in: MedlinePlus