Self-amplifying autocrine actions of BDNF in axon development

Abstract

A critical step in neuronal development is the formation of axon/dendrite polarity, a process involving symmetry breaking in the newborn neuron. Local self-amplifying processes could enhance and stabilize the initial asymmetry in the distribution of axon/dendrite determinants, but the identity of these processes remains elusive. We here report that BDNF, a secreted neurotrophin essential for the survival and differentiation of many neuronal populations, serves as a self-amplifying autocrine factor in promoting axon formation in embryonic hippocampal neurons by triggering two nested positive-feedback mechanisms. First, BDNF elevates cytoplasmic cAMP and protein kinase A activity, which triggers further secretion of BDNF and membrane insertion of its receptor TrkB. Second, BDNF/TrkB signaling activates PI3-kinase that promotes anterograde transport of TrkB in the putative axon, further enhancing local BDNF/TrkB signaling. Together, these self-amplifying BDNF actions ensure stable elevation of local cAMP/protein kinase A activity that is critical for axon differentiation and growth.

Fig. 1.

Secreted BDNF exerts autocrine actions on axon differentiation and growth. (A) Images of hippocampal neurons in 2-d culture, immunostained with axon marker Tau-1 and neuron marker Tuj-1. Cells were incubated with medium containing IgG-Fc (20 μg/ml), TrkB-Fc (20 μg/ml), or BDNF antibodies (5 μg/ml) or transfected with BDNF-siRNA (together with GFP). Arrowhead, axon; arrow: siRNA-transfected cell; *, untransfected cell. (B) Phenotype of 2-d neurons after treatments similar to those in A, including untreated cells (control 1), and untransfected cells in the same cultures (control 2) as those BDNF-siRNA-transfected cells. siRNA-SC, scrambled form of siRNA; SA, single axon; NA, no axon; MA, multiple axons. Data presented as average SEM (n > 50 cells/culture, 3–5 cultures each; **P < 0.01, Tukey test). (C) Composite tracing of axons from 25 randomly sampled neurons from 2-d cultures that were not treated (control 1), treated with BDNF (50 ng/ml), TrkB-Fc (20 μg/ml), K252a (100 nM), K252b (100 nM), or KT5720 (2 μM), or BDNF-siRNA. (D) Axon/dendrite growth in cultures treated with indicated chemicals for 24–36 h (began at 12 h after plating). Data presented as average axon or dendrite length (SEM, n > 50 cells/culture, 3–5 cultures each). *Data significantly different from untreated neurons (control 1) or untransfected neurons in the same culture (control 2) are marked by (P < 0.05, t test).

Fig. 2.

Secretion of BDNF-pHluorin triggered by elevating cAMP or applying BDNF. (A) Fluorescence images of stage 2 (unpolarized, A1) and stage 3 (polarized, A2) hippocampal neurons expressing pH-sensitive fusion protein BDNF-pHluorin, before and at different times after exposure to Sp-cAMPS or forskolin. Insets: Neurite or axon/dendrite tips where pHluorin fluorescence was measured and coded in pseudo colors linearly by the scale shown on the left. (B) Average traces depicting relative pHluorin fluorescence intensity before and after treatment with forskolin and Sp-cAMPS, in the absence or presence of KT7250, for stage 2 and 3 neurons, with the intensity normalized by the mean intensity during the last 3 min before the treatment. n, number of neurons measured. (C and D) Similar to A and B, except that recombinant BDNF (50 ng/mL) was applied.

Fig. 3.

BDNF-induced cAMP elevation and axon differentiation. (A) BDNF induced an elevation of cAMP. (A1), Hippocampal neurons at stage 2 (S2, unpolarized) and 3 (S3, polarized), transfected with FRET indicator for cAMP (ICUE; see SI Materials and Methods). Shown are images of CFP fluorescence and FRET signals at different times after bath-applying of BDNF (50 ng/ml) or contact with a BDNF-coated bead. The FRET signal is the ratio of CFP to YFP fluorescence [F(CFP)/F(YFP)], measured in the boxed regions. Bar, 20 μm. (A2), Traces of cAMP changes at the neurite (or axon/dendrite) tip of individual neurites (normalized by the mean values for 3 min prior to BDNF). (A3) Summary of cAMP changes at the neurite (or axon/dendrite) tip, shown by FRET signals (normalized as in A2), in the absence or presence of K252a (or K252b). Error bar = SEM (n = 5–10 cells each, 1 or 2 neurites/cell). (B) Axon development triggered by local exposure to BDNF or forskolin-induced cAMP elevation. (B1), Images showing local contact of a single BDNF-coated bead with an undifferentiated neurite (10 h after plating, Left), which developed into an axon (arrow heads) 48 h later, as shown by Tau-1 staining (Right). (B2), Percentage (SEM, n > 15 each, *P < 0.05, t test) of neurites that differentiated into axon or dendrite, or retracted at 36–48 h after the contact with a bead coated with BSA (control), forskolin, or BDNF.

Fig. 4.

Redistribution of TrkB receptor and cAMP toward axon after polarization. (A) Surface TrkB and cytoplasmic cAMP level in stage-2 and stage-3 neurons. (A1) Hippocampal neurons immunostained with ectodomain-specific antibodies for surface TrkB receptors (green). Cell surface was costained with R-ConA (red) for membrane area normalization. (Scale bar, 20 μm.) (A2) FRET images of stage-2 (or stage-3) neurons cotransfected with the FRET indictor for PKA activity (AKAR). (B) Summary graph of stage-2 (B1) or stage-3 (B2) neurons showing mean fluorescence intensity of surface TrkB (±SEM, n = 35–50; **P < 0.01, Tukey test, normalized by ConA staining, left axis) and mean basal PKA activity, as indicated by the AKAR signal (±SEM, n = 10–35; *P < 0.05, Tukey test, right axis) along neurite (Top) or at 5 μm distal of the neurite. The first five neurites of each cell with highest intensities were compared and ranked from left to right by intensity and averaged among all cells. The average lengths of the intensity-ranked neurites are shown below.

Fig. 5.

Nested positive-feedback mechanisms in autocrine BDNF/TrkB signaling. First, local elevation of cAMP/PKA activity resulting from intrinsic or extrinsic signals causes local secretion, which in turn further elevates local cAMP/PKA activity. Second, local cAMP/PKA activity causes local surface insertion of TrkB, further amplifying BDNF/TrkB signaling and enhancing local cAMP/PKA activity. Third, elevating BDNF/TrkB signaling activates PI3K activity, which promotes anterograde trafficking of TrkB, further enhancing local surface insertion of TrkB and BDNF/TrkB signaling.