Article Figures & Data
Figures
- Figure 2.
ERK5 is activated in newly generated granule neurons and is necessary for BDNF-mediated survival. A, BDNF activates ERK5 in newly generated granule neurons. Granule neurons were deprived of survival factors and left untreated or treated with BDNF, as in Figure 1 B, for the indicated periods. Immunoblotting was done with a phospho-ERK5 antibody that recognizes ERK5 that is phosphorylated within the TEY motif (top; BioSource) or with an antibody that recognizes ERK5, regardless of phosphorylation state (bottom; Calbiochem). B, BDNF stimulates ERK5 kinase activity. Granule neurons (P6 + 5DIV) were deprived of survival factors and left untreated or treated with BDNF (100 ng/ml) for 15 min. Immunoprecipitated ERK5 was subjected to an in vitro kinase assay using GST-MEF2A as substrate. C, BDNF induces ERK5 phosphorylation in newly generated, but not in mature, cerebellar granule neurons. Newly generated (P6 + 4DIV or + 5DIV) or mature (P6 + 8DIV) granule neurons were treated with BDNF, as in Figure 1 B, for 30 min. Immunoblotting was done with the phospho-ERK5 antibody (top), the ERK5 antibody (second panel), the phospho-Akt antibody (third panel), and the Akt antibody (bottom). D, ERK5 mediates BDNF-induced granule neuron survival. Cerebellar granule neurons (P6 + 5DIV) were transfected with a dominant-negative MEK5 (MEK5AA) or its control vector together with an expression plasmid encoding β-galactosidase. Transfected cultures were deprived of survival factors and left untreated or treated with BDNF (100 ng/ml). After 2 d, cells were fixed and analyzed for cell survival and death. Percentage of survival is shown as mean ± SEM. BDNF significantly increased survival of vector-transfected cerebellar granule neurons (ANOVA; p < 0.001; n = 3) but not MEK5AA-expressing granule neurons. To determine the specificity of MEK5AA in granule neurons, we tested the effect of MEK5AA on BDNF-induced phosphorylation of ERK1/2 immunocytochemically using the phosphoERK1/2 antibody. In two experiments, nearly 80% of vector-transfected, BDNF-treated granule neurons displayed phospho ERK1/2 immunoreactivity. The expression of a dominant interfering form of MEK1 (MEK1KA97) reduced the number of phosphoERK1/2-positive cells to 37%. However, the expression of MEK5AA failed to reduce BDNF activation of ERK1/2, because 77% of MEK5AA-expressing granule neurons were phosphoERK1/2 positive.
- Figure 6.
NT-3 is required for BDNF-induced survival of newly generated cerebellar granule neurons. A, Newly generated cerebellar granule neurons (P6 + 5DIV) were deprived of survival factors and left untreated or treated with BDNF (100 ng/ml; Control) or together with a mouse monoclonal neutralizing antibody specific to NT-3 (anti-NT-3; 200 ng/ml; R&D) or with a control neutralizing antibody specific to ciliary neurotrophic factor (anti-CNTF; 200 ng/ml; R&D). Two days later, cultures were fixed and analyzed for cell survival and death. Percentage of survival is shown as mean ± SEM. BDNF significantly promoted the survival of granule neurons in control or anti-CNTF-treated cultures (ANOVA; p < 0.001; n = 3) but not in the presence of the anti-NT-3 antibody. B, C, NT-3 is necessary for BDNF-mediated neuron survival. Cultures of newly generated cerebellar granule neurons (P5 + 1DIV) from mice with disruption of the NT-3 gene generated by CRE-mediated recombination of floxed NT-3 alleles were deprived of survival factors and left untreated or supplemented with increasing amounts of BDNF (0, 25, or 50 ng/ml). After 3 d, cultures were fixed and analyzed for cell survival and death. Shown in B are percentages of apoptosis for matched wild-type littermates (nt3+/+; closed symbols) and knock-out littermates (nt3-/-; open symbols). Shown in C is mean percentage of reduction in apoptosis ± SEM for 25 ng/ml (25) or 50 ng/ml (50) BDNF relative to untreated cultures. The survival effect of BDNF is significantly reduced in NT-3 knock-out (nt3-/-) cultures relative to wild-type controls (nt3+/+) (ANOVA; p < 0.01; n = 3). D, Exogenous NT-3 rescues BDNF-mediated neuron survival in NT-3 knock-out cells. Cultures of newly generated cerebellar granule neurons (P5 + 1DIV) were prepared from nt3+/+ or nt3-/- mice and were deprived of survival factors or treated with 50 ng/ml BDNF alone (BDNF) or 50 ng/ml BDNF plus 50 ng/ml NT-3 (BDNF+NT-3). After 3 d, granule neuron cultures were fixed and analyzed for cell survival and death. Shown is mean percentage of reduction in apoptosis ± SEM relative to untreated cultures. The addition of NT-3 together with BDNF led to a significant increase in survival of NT-3 knock-out (nt3-/-) granule neurons relative to BDNF alone (ANOVA; p < 0.001; n = 4) but not of nt3+/+ granule neurons.
- Figure 1.
BDNF promotes survival of cerebellar granule neurons in a temporally defined manner. A, Newly generated (P6 + 3DIV) or mature (P6 + 7DIV) cerebellar granule neurons were deprived of survival factors and left untreated (control) or treated with BDNF (100 ng/ml) or with full survival medium containing 30 mm KCl and 10% serum (conditioned medium, CM). After 48 hr, cultures were fixed and subjected to immunofluorescence. Percentage of apoptosis is shown as mean ± SEM. BDNF significantly reduced apoptosis in newly generated cerebellar granule neurons (ANOVA; p < 0.0005; n = 3) but had no effect on apoptosis of mature granule neurons. Full survival medium reduced apoptosis of both newly generated and mature cerebellar granule neurons (ANOVA; p < 0.0001; n = 3). B,C, BDNF induces the phosphorylation of ERK1/2 and Akt in both newly generated and mature cerebellar granule neurons. Newly generated (P6 + 3DIV) and mature (P6 + 7DIV) cultures of cerebellar granule neurons were deprived of survival factors for 1 hr and then treated with BDNF (100 ng/ml) for the indicated periods. In B, immunoblotting was performed with an antibody that specifically recognizes ERK1/2 when phosphorylated within the TEY motif (B, top; Promega) or with an antibody that recognizes ERK1/2, regardless of the phosphorylation state (B, bottom; New England Biolabs). In C, immunoblotting was performed with an antibody that recognizes Akt when phosphorylated at serine 478 (C, top; New England Biolabs) or with an antibody that recognizes Akt, regardless of phosphorylation state (C, bottom; New England Biolabs).
- Figure 3.
MEF2 mediates BDNF-induced survival of newly generated cerebellar granule neurons. A, Schematic representation of dominant-negative MEF2s. In MEF2CR24L, arginine 24 is substituted to lysine within the conserved DNA-binding domain. In MEF2A-TA, the key regulatory sites of phosphorylation threonines 312 and 319 are replaced by alanines. B, MEF2 mediates BDNF-induced granule neuron survival. Granule neurons were transfected with an expression plasmid encoding the dominant interfering forms of MEF2 (see Fig.3A) or the control vector together with the β-galactosidase expression plasmid. Transfected cultures were analyzed as in Figure 2 D. BDNF significantly increased survival of vector-transfected cerebellar granule neurons (ANOVA; p < 0.001; n = 3) but not of MEF2CR24L- or MEF2A-TA-expressing granule neurons. C, Knockdown of MEF2A inhibits BDNF-induced granule neuron survival. Granule neurons (P6 + 2DIV) were transfected with the U6 control vector or the U6/MEF2A plasmid encoding MEF2A hairpin RNAs, together with the β-galactosidase expression plasmid. After 2 d, cells were changed to medium without survival factors or were supplemented with BDNF (100 ng/ml). After 1 d, cells were fixed and subjected to immunofluorescence. Percentage of survival is shown as mean ± SEM. BDNF significantly increased survival of U6-transfected cerebellar granule neurons (ANOVA; p < 0.0005; n = 3) but not of MEF2A hpRNA-expressing transfected granule neurons. BDNF-induced survival was significantly reduced in MEF2A hpRNA-expressing neurons (percentage of survival, 16.3 ± 1.8; p < 0.01; ANOVA; n = 3) but not in Cdk2 hpRNA-expressing neurons (percentage of survival, 32.3 ± 4.7; n = 3) when compared with U6-transfected granule neurons (percentage of survival, 40.7 ± 1.5; n = 3).
- Figure 4.
NT-3: a novel target of the BDNF-induced ERK5-MEF2 signaling pathway in newly generated granule neurons. A, B, BDNF induces the expression of NT-3 mRNA in newly generated, but not in mature, cerebellar granule neurons. In A, P6 + 3DIV and P6 + 7DIV cerebellar granule neurons were deprived of survival factors for 4 hr and treated with BDNF for the indicated periods. In B, P6 + 5DIV, P6 + 7DIV, or P6 + 9DIV granule neurons were treated with 10 μg/ml insulin in the presence or absence of BDNF for 24 hr. RNA was isolated and subjected to RT-PCR with primers specific for c-fos (Fig. 4 A only), NT-3, or GAPDH. C, BDNF activates NT-3 promoter-dependent transcription. NT-3-luciferase reporter constructs containing nucleotides -1087 to +91 or nucleotides -838 to +91 relative to the NT-3 transcription start site of the NT-3 promoter were transfected in newly generated cerebellar granule neurons (P6 + 3DIV). After 6 hr, cells were treated without or with BDNF (100 ng/ml) for 20 hr, and luciferase activity was determined. Shown are mean ± SEM luciferase values normalized relative to uninduced -1087/+91 reporter. BDNF significantly induced the -1087 NT-3-luciferase reporter gene (ANOVA; p < 0.0001; n = 5) but not the -838 NT-3-luciferase reporter. D, The NT-3 promoter contains a MEF2-binding site. Extracts from cerebellar granule neurons (CGN; P6 + 3DIV) were incubated in the presence or absence a 30 bp oligonucleotide containing the putative NT-3 MRE, with or without a 10-fold excess of an oligonucleotide containing a consensus MRE (10x WT MRE), an oligonucleotide containing a mutant MRE does not bind MEF2 (10x Mut MRE), or a polyclonal antibody against MEF2A (left). Similar reactions were performed with extracts from 293T cells overexpressing MEF2A (right). FP, Free oligonucleotide probe. E, The MRE is required for BDNF induction of NT-3 transcription in response to BDNF. A NT-3-luciferase reporter gene containing nucleotides -1117 to +91 of the NT-3 promoter with either the wild-type MRE (WT MRE) or a point mutation of the MRE (Mut MRE) that abolished MEF2 binding in vitro were introduced to P6 + 3DIV granule neurons, as in C. After transfection, cells were analyzed as in C. BDNF induced the wild-type -1117 NT-3-luciferase reporter significantly (ANOVA; p < 0.0001; n = 5) but not the Mut MRE -1117 NT-3-luciferase reporter gene. F, Granule neurons were transfected with the -1087 NT-3-luciferase reporter gene together with an expression plasmid encoding MEF2A-TA or MEF2CR24L or the control plasmid. Transfected cultures were analyzed as in Figure 4C. BDNF induced the NT-3 promoter significantly in control-transfected cultures (ANOVA; p < 0.0005; n = 3) but not in MEF2A-TA- or MEF2CR24L-expressing granule neurons. G, Activation of the ERK5 pathway is required for BDNF-induced NT-3 transcription. Newly generated granule neurons (P6 + 3DIV) were transfected, as in C, with the -1087 NT-3-luciferase reporter gene and with an expression vector encoding dominant-negative MEK5 (MEK5AA) or its control vector and analyzed as in C. BDNF significantly induced the NT-3 reporter in vector-transfected cultures (ANOVA; p < 0.005; n = 4) but not in MEK5AA-expressing granule neurons.
- Figure 5.
MEF2 cooperates with CREB to mediate BDNF-induced NT-3 transcription. A, The NT-3 promoter contains a CREB-binding sequence. Extracts from cerebellar granule neurons (CGN; P6 + 3DIV) were incubated in the presence or absence a 30 bp oligonucleotide containing the putative NT-3 CRE, with or without a 10-fold excess of the wild-type oligonucleotide (10x WT CRE), an oligonucleotide containing a mutant CRE (10x Mut CRE), or a polyclonal antibody against either CREB or MEF2A. FP, Free oligonucleotide probe. B, The CRE is required for BDNF-induction of NT-3 transcription in response to BDNF. An NT-3-luciferase reporter gene containing nucleotides -1087 to +91 of the NT-3 promoter with either the wild-type CRE (WT) or a point mutation of the CRE (Mut CRE) that abolished CREB binding in vitro was introduced to P6 + 3DIV granule neurons and analyzed as in Figure 4C. BDNF induced the wild-type -1087 NT-3-luciferase reporter significantly (ANOVA; p < 0.005; n = 6) but not the Mut CRE -1087 NT-3-luciferase reporter gene. C, Dominant-negative CREB prevents BDNF induction of NT-3. Granule neurons were transfected with the -1087 NT-3-luciferase reporter gene together with an expression plasmid encoding CREBM1 or a control plasmid. Transfected cultures were analyzed as in Figure 4C. BDNF induced the NT-3 promoter significantly in control-transfected cultures (ANOVA; p < 0.0005; n = 3) but not in CREBM1-expressing granule neurons. D, The ERK1/2 pathway is necessary for BDNF-induced NT-3 transcription. Newly generated granule neurons (P6 + 3DIV) were transfected as in Figure 4C with the -1087 NT-3-luciferase reporter gene and an expression vector encoding dominant-negative MEK1 (MEK1KA97) or its control vector and analyzed as in Figure 4C. BDNF significantly induced the NT-3 reporter in vector-transfected cultures (ANOVA; p < 0.05; n = 3) but not in MEK1KA97-expressing granule neurons.
