Neuronal release of proBDNF

Abstract

Pro-brain-derived neurotrophic factor (proBDNF) and mature BDNF utilize distinct receptors to mediate divergent neuronal actions. Using new tools to quantitate endogenous BDNF isoforms, we found that mouse neurons secrete both proBDNF and mature BDNF. The highest levels of proBDNF and p75 were observed perinatally and declined, but were still detectable, in adulthood. Thus, BDNF actions are developmentally regulated by secretion of proBDNF or mature BDNF and by local expression of p75 and TrkB.

Figure 1

Detection of proBDNF and mature BDNF in mouse hippocampus and secretion of proBDNF and mature BDNF by cultured hippocampal neurons. (a) Hippocampal lysates of 4-week-old mice of the indicated genotype were immunoprecipitated with antibody to HA and immunoblotted as indicated. mAb287 is the monoclonal antibody to the BDNF prodomain that we characterized in Supplementary Figure 1. (b,c) Neurons were cultured from P0 mice of the indicated genotype for 7 d in the presence (b) or absence (c) of alpha 2 anti-plasmin. Media (M) and cell lysates (L) were immmunoprecipitated (IP) with antibody to HA. proBDNF and mature BDNF were detected with antibody to HA.11. (d,e) Neuronal cultures were established as in b. We added TrkB-Fc (d) or TrkB-Fc or TrkA-Fc (e) to the media 3 d before harvest. Neurons cultured in the absence of TrkB-Fc were analyzed as controls. Lysates were immunoprecipitated; Fc proteins were captured by Protein A-Sepharose (capture). (f) Embryonic day 16.5 (E16.5) neurons were cultured as in b and stimulated with 56 mM KCl for 90 min. The media and cell lysates were processed as indicated. (g) E16.5 hippocampal neurons were cultured as described previously, with or without fluorouracil for 2 d. At 7 d in vitro, cells were stimulated with 56 mM KCl for 90 min, and immunoprecipitation/western blot analysis was carried out as indicated. All of our animal studies were approved by the Institutional Animal Care and Use Committee of Weill Cornell Medical College.

Figure 2

Developmental regulation of proBDNF and p75 expression. (a) Immunofluorescence detection of BDNF in mouse hippocampi (P28) using mAb287, antibodies to HA and Cy3-conjugated streptavidin, shown in serial sections of Bdnf-HA/+ or wild-type brain. Non-immune mouse IgG was used as a control. Scale bars represent 250 µm. (b) Hippocampal lysates from mice of the indicated genotype and ages were subjected to immunoprecipitation/western blot analysis for BDNF isoforms (top), probed to detect p75 (middle) or probed for ERK as a loading control (bottom). (e) Immunofluorescence detection of proBDNF or p75 using 1-week-old and 4-week-old mouse brains. Non-immune IgG was used as a control. Scale bars represent 250 µm.