Limits...
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.

Show MeSH

Related in: MedlinePlus

Internalization of the pro-BDNF–p75NTR complex in cultured astrocytes. (A) Time sequence of pro-BDNF–QD–p75-GFP internalization in cultured astrocytes by TIRF imaging. White arrowheads indicate pro-BDNF–QDs (red) in close proximity to the membrane of an astrocyte transfected with p75-GFP (green). Yellow arrowheads point to reference QDs. Insets depict p75-GFP fluorescence that concentrates at the site of the QD. Bar, 5 μm. (B) Pro-BDNF–QD internalization in astrocytes transfected with p75-GFP (nine cells) or Lck-GFP (six cells). (C) Pro-BDNF–QD internalization in astrocytes from p75NTR+/+ (22 cells) and p75NTR−/− (11 cells) mice. (D) Immunocytochemistry showing colocalization (arrowheads) between QDs (blue) and p75NTR (red) in astrocytes transfected with p75-GFP or Lck-GFP (green). Bar, 2 μm. Data are means ± SEM (error bars). *, P ≤ 0.05.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2568011&req=5

fig3: Internalization of the pro-BDNF–p75NTR complex in cultured astrocytes. (A) Time sequence of pro-BDNF–QD–p75-GFP internalization in cultured astrocytes by TIRF imaging. White arrowheads indicate pro-BDNF–QDs (red) in close proximity to the membrane of an astrocyte transfected with p75-GFP (green). Yellow arrowheads point to reference QDs. Insets depict p75-GFP fluorescence that concentrates at the site of the QD. Bar, 5 μm. (B) Pro-BDNF–QD internalization in astrocytes transfected with p75-GFP (nine cells) or Lck-GFP (six cells). (C) Pro-BDNF–QD internalization in astrocytes from p75NTR+/+ (22 cells) and p75NTR−/− (11 cells) mice. (D) Immunocytochemistry showing colocalization (arrowheads) between QDs (blue) and p75NTR (red) in astrocytes transfected with p75-GFP or Lck-GFP (green). Bar, 2 μm. Data are means ± SEM (error bars). *, P ≤ 0.05.

Mentions: To obtain further insight into the possible regulation of pro-BDNF uptake in astrocytes, total internal reflection fluorescence (TIRF) microscopy (Thompson and Steele, 2007) was used to visualize the formation of single endocytic vesicles in real time (Fig. 3 A and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200806137/DC1). Cultured astrocytes were transfected with p75NTR tagged with GFP (p75-GFP) for evanescent light excitation of p75-GFP residing within or in close proximity to the plasma membrane. The binding of pro-BDNF to p75-GFP was imaged in real time using pro-BDNF immunocomplexed with 10 nM of quantum dots (QDs; Fig. S3). Once a pro-BDNF–QD was found in the vicinity of the plasma membrane of a p75-GFP–expressing astrocyte, p75-GFP fluorescence became concentrated at the site of the QD within a few seconds, presumably reflecting the formation of endocytic vesicles and internalization of the pro-BDNF–QDs. Confocal microscopy (Fig. 3 B) showed that internalization of pro-BDNF–QDs was inhibited at a nonpermissive temperature (ice cold) for endocytosis and was restored by raising the temperature to 37°C for 10–20 min. QD uptake also depended on the level of p75NTR expression: although astrocytes overexpressing p75-GFP showed high levels of QD internalization, significantly fewer QDs were taken up in astrocytes transfected with plasma membrane–linked GFP (Lck-GFP), which only relies on endogenous p75NTR for internalization (Fig. 3, B and D). Likewise, QD uptake was virtually abolished in astrocytes prepared from p75NTR−/− mice (Fig. 3 C). Moreover, QD internalization required prior coupling to pro-BDNF, as it ceased when the α–pro-BDNF antibody was omitted from the immunocomplexes for control (Fig. 3 B). Lastly, internalized QDs colocalized with clathrin and EEA1 (Fig. S3), confirming in primary cultures the mechanism of pro-BDNF endocytosis shown in stimulated slices.


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)

Internalization of the pro-BDNF–p75NTR complex in cultured astrocytes. (A) Time sequence of pro-BDNF–QD–p75-GFP internalization in cultured astrocytes by TIRF imaging. White arrowheads indicate pro-BDNF–QDs (red) in close proximity to the membrane of an astrocyte transfected with p75-GFP (green). Yellow arrowheads point to reference QDs. Insets depict p75-GFP fluorescence that concentrates at the site of the QD. Bar, 5 μm. (B) Pro-BDNF–QD internalization in astrocytes transfected with p75-GFP (nine cells) or Lck-GFP (six cells). (C) Pro-BDNF–QD internalization in astrocytes from p75NTR+/+ (22 cells) and p75NTR−/− (11 cells) mice. (D) Immunocytochemistry showing colocalization (arrowheads) between QDs (blue) and p75NTR (red) in astrocytes transfected with p75-GFP or Lck-GFP (green). Bar, 2 μm. Data are means ± SEM (error bars). *, P ≤ 0.05.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2568011&req=5

fig3: Internalization of the pro-BDNF–p75NTR complex in cultured astrocytes. (A) Time sequence of pro-BDNF–QD–p75-GFP internalization in cultured astrocytes by TIRF imaging. White arrowheads indicate pro-BDNF–QDs (red) in close proximity to the membrane of an astrocyte transfected with p75-GFP (green). Yellow arrowheads point to reference QDs. Insets depict p75-GFP fluorescence that concentrates at the site of the QD. Bar, 5 μm. (B) Pro-BDNF–QD internalization in astrocytes transfected with p75-GFP (nine cells) or Lck-GFP (six cells). (C) Pro-BDNF–QD internalization in astrocytes from p75NTR+/+ (22 cells) and p75NTR−/− (11 cells) mice. (D) Immunocytochemistry showing colocalization (arrowheads) between QDs (blue) and p75NTR (red) in astrocytes transfected with p75-GFP or Lck-GFP (green). Bar, 2 μm. Data are means ± SEM (error bars). *, P ≤ 0.05.
Mentions: To obtain further insight into the possible regulation of pro-BDNF uptake in astrocytes, total internal reflection fluorescence (TIRF) microscopy (Thompson and Steele, 2007) was used to visualize the formation of single endocytic vesicles in real time (Fig. 3 A and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200806137/DC1). Cultured astrocytes were transfected with p75NTR tagged with GFP (p75-GFP) for evanescent light excitation of p75-GFP residing within or in close proximity to the plasma membrane. The binding of pro-BDNF to p75-GFP was imaged in real time using pro-BDNF immunocomplexed with 10 nM of quantum dots (QDs; Fig. S3). Once a pro-BDNF–QD was found in the vicinity of the plasma membrane of a p75-GFP–expressing astrocyte, p75-GFP fluorescence became concentrated at the site of the QD within a few seconds, presumably reflecting the formation of endocytic vesicles and internalization of the pro-BDNF–QDs. Confocal microscopy (Fig. 3 B) showed that internalization of pro-BDNF–QDs was inhibited at a nonpermissive temperature (ice cold) for endocytosis and was restored by raising the temperature to 37°C for 10–20 min. QD uptake also depended on the level of p75NTR expression: although astrocytes overexpressing p75-GFP showed high levels of QD internalization, significantly fewer QDs were taken up in astrocytes transfected with plasma membrane–linked GFP (Lck-GFP), which only relies on endogenous p75NTR for internalization (Fig. 3, B and D). Likewise, QD uptake was virtually abolished in astrocytes prepared from p75NTR−/− mice (Fig. 3 C). Moreover, QD internalization required prior coupling to pro-BDNF, as it ceased when the α–pro-BDNF antibody was omitted from the immunocomplexes for control (Fig. 3 B). Lastly, internalized QDs colocalized with clathrin and EEA1 (Fig. S3), confirming in primary cultures the mechanism of pro-BDNF endocytosis shown in stimulated slices.

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