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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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Vesicles containing pro-BDNF–p75NTR express the Vamp2 component of the SNARE core complex for vesicle fusion. (A) Colocalization between GFAP, pro-BDNF, and Vamp2 immunoreactivity in astrocytes 10 min after TBS. Colocalization signal (arrowheads) is shown at the site of astrocytic contact with a neuron (box and inset 1) and astrocytic processes (box and inset 2). Bar, 20 μm. (B) Immunocytochemistry showing colocalization between pro-BDNF–QDs and Vamp2 in astrocytes transfected with p75-GFP. Right panels depict QD/Vamp2 colocalization (arrowheads) in a selected astrocytic area (boxed area). Bar, 2 μm. (C) Western blot showing p75NTR and Vamp2 expression in endocytic vesicles immunopurified (IP) by magnetic beads coated with α-p75NTR, α-Vamp2, or α-Map2 from astrocytes untreated or treated with BDNF (mix).
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fig5: Vesicles containing pro-BDNF–p75NTR express the Vamp2 component of the SNARE core complex for vesicle fusion. (A) Colocalization between GFAP, pro-BDNF, and Vamp2 immunoreactivity in astrocytes 10 min after TBS. Colocalization signal (arrowheads) is shown at the site of astrocytic contact with a neuron (box and inset 1) and astrocytic processes (box and inset 2). Bar, 20 μm. (B) Immunocytochemistry showing colocalization between pro-BDNF–QDs and Vamp2 in astrocytes transfected with p75-GFP. Right panels depict QD/Vamp2 colocalization (arrowheads) in a selected astrocytic area (boxed area). Bar, 2 μm. (C) Western blot showing p75NTR and Vamp2 expression in endocytic vesicles immunopurified (IP) by magnetic beads coated with α-p75NTR, α-Vamp2, or α-Map2 from astrocytes untreated or treated with BDNF (mix).

Mentions: How is the endocytic pro-BDNF recycled for exocytosis? One potential mechanism making endocytic vesicles available for secretion involves the molecular machinery deputed to exocytic fusion. Astrocytes are known to express components of the core SNARE complex, including Vamp2 (Montana et al., 2006). Colocalization of pro-BDNF with Vamp2 was detected within astrocytes in slices 10 min after TBS (Fig. 5 A) or in cultured astrocytes transfected with p75-GFP and exposed to pro-BDNF–QDs (Fig. 5 B). The expression of Vamp2 on BDNF-containing vesicles was confirmed by Western blot analysis of endocytic vesicles purified by magnetic beads coated with α-p75NTR antibodies (Fig. 5 C). Conversely, endocytic vesicles purified using beads coated with α-Vamp2 antibodies were immunoreactive for p75NTR. Interestingly, treatment with BDNF (mix) for 10 min enhanced the recovery of vesicles expressing both Vamp2 and p75NTR. Beads coated with antibodies against the neuronal marker microtubule-associated protein 2 (Map2) were used as a control. These data indicate that endocytic vesicles expressing p75NTR may represent the main storage compartment for endocytosed pro-BDNF before routing to the secretory pathway. This process might take place either by recycling of pro-BDNF–p75NTR complexes to the surface or by pro-BDNF recycling upon its dissociation from p75NTR. Moreover, given that TeNT prevented BDNF secretion (Fig. 4 F), all of these data indicate that after endocytosis in astrocytes, vesicles containing the neurotrophin may undergo regulated recycling via a SNARE-dependent mechanism.


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)

Vesicles containing pro-BDNF–p75NTR express the Vamp2 component of the SNARE core complex for vesicle fusion. (A) Colocalization between GFAP, pro-BDNF, and Vamp2 immunoreactivity in astrocytes 10 min after TBS. Colocalization signal (arrowheads) is shown at the site of astrocytic contact with a neuron (box and inset 1) and astrocytic processes (box and inset 2). Bar, 20 μm. (B) Immunocytochemistry showing colocalization between pro-BDNF–QDs and Vamp2 in astrocytes transfected with p75-GFP. Right panels depict QD/Vamp2 colocalization (arrowheads) in a selected astrocytic area (boxed area). Bar, 2 μm. (C) Western blot showing p75NTR and Vamp2 expression in endocytic vesicles immunopurified (IP) by magnetic beads coated with α-p75NTR, α-Vamp2, or α-Map2 from astrocytes untreated or treated with BDNF (mix).
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Related In: Results  -  Collection

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fig5: Vesicles containing pro-BDNF–p75NTR express the Vamp2 component of the SNARE core complex for vesicle fusion. (A) Colocalization between GFAP, pro-BDNF, and Vamp2 immunoreactivity in astrocytes 10 min after TBS. Colocalization signal (arrowheads) is shown at the site of astrocytic contact with a neuron (box and inset 1) and astrocytic processes (box and inset 2). Bar, 20 μm. (B) Immunocytochemistry showing colocalization between pro-BDNF–QDs and Vamp2 in astrocytes transfected with p75-GFP. Right panels depict QD/Vamp2 colocalization (arrowheads) in a selected astrocytic area (boxed area). Bar, 2 μm. (C) Western blot showing p75NTR and Vamp2 expression in endocytic vesicles immunopurified (IP) by magnetic beads coated with α-p75NTR, α-Vamp2, or α-Map2 from astrocytes untreated or treated with BDNF (mix).
Mentions: How is the endocytic pro-BDNF recycled for exocytosis? One potential mechanism making endocytic vesicles available for secretion involves the molecular machinery deputed to exocytic fusion. Astrocytes are known to express components of the core SNARE complex, including Vamp2 (Montana et al., 2006). Colocalization of pro-BDNF with Vamp2 was detected within astrocytes in slices 10 min after TBS (Fig. 5 A) or in cultured astrocytes transfected with p75-GFP and exposed to pro-BDNF–QDs (Fig. 5 B). The expression of Vamp2 on BDNF-containing vesicles was confirmed by Western blot analysis of endocytic vesicles purified by magnetic beads coated with α-p75NTR antibodies (Fig. 5 C). Conversely, endocytic vesicles purified using beads coated with α-Vamp2 antibodies were immunoreactive for p75NTR. Interestingly, treatment with BDNF (mix) for 10 min enhanced the recovery of vesicles expressing both Vamp2 and p75NTR. Beads coated with antibodies against the neuronal marker microtubule-associated protein 2 (Map2) were used as a control. These data indicate that endocytic vesicles expressing p75NTR may represent the main storage compartment for endocytosed pro-BDNF before routing to the secretory pathway. This process might take place either by recycling of pro-BDNF–p75NTR complexes to the surface or by pro-BDNF recycling upon its dissociation from p75NTR. Moreover, given that TeNT prevented BDNF secretion (Fig. 4 F), all of these data indicate that after endocytosis in astrocytes, vesicles containing the neurotrophin may undergo regulated recycling via a SNARE-dependent mechanism.

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