A retrograde neuronal survival response: target-derived neurotrophins regulate MEF2D and bcl-w

Abstract

Survival and maturation of dorsal root ganglia sensory neurons during development depend on target-derived neurotrophins. These target-derived signals must be transmitted across long distances to alter gene expression. Here, we address the possibility that long-range retrograde signals initiated by target-derived neurotrophins activate a specialized transcriptional program. The transcription factor MEF2D is expressed in sensory neurons; we show that expression of this factor is induced in response to target-derived neurotrophins that stimulate the distal axons. We demonstrate that MEF2D regulates expression of an anti-apoptotic bcl-2 family member, bcl-w. Expression of mef2d and bcl-w is stimulated in response to activation of a Trk-dependent ERK5/MEF2 pathway, and our data indicate that this pathway promotes sensory neuron survival. We find that mef2d and bcl-w are members of a larger set of retrograde response genes, which are preferentially induced by neurotrophin stimulation of distal axons. Thus, activation of an ERK5/MEF2D transcriptional program establishes and maintains the cellular constituents of functional sensory circuits.

Figure 1.

Neurotrophins induce mef2d expression in DRG neurons in response to distal axon stimulation. A, MEF2D protein is detected in vivo in dorsal root ganglia at E18 (lanes 1 and 2 represent 2 separate animals). As a positive control, lysates of neurons over-expressing MEF2VP16 were blotted for MEF2D. B, MEF2D immunostaining of dorsal root ganglia at P0 in vivo. Scale bar, 10 μm. C, Expression of mef2d mRNA in DRG neurons in response to neurotrophin (NT) stimulation. DRG neurons were treated with neurotrophins (100 ng/ml NGF plus BDNF) for 2 h either on distal axons (DA) or on cell bodies (CB). Mef2D mRNA expression is specifically induced by distal axon stimulation and not by cell body stimulation. Expression is compared with DRG neurons treated with vehicle (100 ng/ml BSA). Results represent the mean ± SEM of eight experiments, *p < 0.05. D, Mef2d mRNA expression is upregulated during stimulation of distal axons with NGF alone, BDNF alone, or NGF plus BDNF (NT). Induction of cfos mRNA is shown as a positive control. *p < 0.05. E, MEF2D protein levels were analyzed by Western blotting before and after NT stimulation for 2 h. Normalized relative band density of MEF2D/actin reveal an increase of 21 ± 7% in response to distal axon stimulation.

Figure 2.

MEF2D is required for survival responses to neurotrophins. A, DRG neurons in culture were infected with lentivirus expressing mef2d–GFP RNAi and cell lysates analyzed by Western blotting with indicated antibodies. Mef2d RNAi (+) results in decreased expression of MEF2D protein levels, whereas a control lentivirus (−) does not. Cerebellar lysate (Cb lysate) was run as a positive control. B, Apoptosis of DRG neurons was analyzed by TUNEL staining. Mef2d RNAi reduces survival of DRG neurons in response to neurotrophins when applied to cell bodies (CB) and blocks survival when applied to distal axons (DA). Results represent the mean ± SEM of five experiments, *p < 0.05. C, Representative images of DRG neuron cell bodies grown in culture and stimulated with neurotrophins or vehicle control on cell bodies or on distal axons. Cells with (mef2d RNAi) or without (control) mef2d reduction are shown. Panels show TUNEL staining (red) and DAPI-stained nuclei in control (top) and in neurons expressing mef2d RNAi (bottom).

Figure 3.

ERK5 regulates MEF2D and promotes survival in response to target derived neurotrophins. A, MEF2D is phosphorylated in response to ERK5 activation. COS cells were transfected with CA–MEK5 and WT–ERK5, or WT–ERK5 alone, and lysates were blotted with an antibody to phospho-MEF2 (p-MEF2). B, MEF2D is phosphorylated in response to cell body stimulation (20 ± 8% increase, p < 0.05) for 20 min. Lysates were blotted with an antibody to phospho-MEF2 and with an antibody to pan-actin as a loading control. C, MEF2D is phosphorylated in response to distal axon stimulation (17 ± 6% increase, p < 0.05) for 2 h. Lysates were blotted with an antibody to phospho-MEF2 and with an antibody to pan-actin as a loading control. D, Neurons were infected with an adenovirus that expresses HA-tagged ERK5-dominant negative form (DN–ERK5), then treated with neurotrophins for 30 min. Compared with uninfected neurons, expression of DN–ERK5 prevents ERK5 phosphorylation without inhibiting ERK1/2 activation. E, ERK5 supports neuronal survival induced by target-derived neurotrophins. Cell apoptosis was measured by TUNEL staining in uninfected neurons and in neurons infected with WT–ERK5 or DN–ERK5 for 2 d. DN–ERK5 blocks survival of neurons that depend on target-derived neurotrophins, whereas DN–ERK5 has a lesser effect on survival supported by cell body stimulation. All data shown are means ± SEM, *p ≤ 0.05.

Figure 4.

Target-derived neurotrophins induce bcl-w and mef2d expression in compartmented cultures by a Trk-dependent ERK5/MEF2 pathway. A, Expression of bcl-w mRNA in response to neurotrophin stimulation. DRG neurons were treated with neurotrophin (NT = 100 ng/ml NGF plus BDNF) for 2 h either on distal axons (DA) or on cell bodies (CB). Bcl-w mRNA expression is specifically induced by distal axon stimulation and not by cell body stimulation. Bcl-w mRNA is also upregulated during distal axon stimulation with NGF or BDNF alone. All data show mean ± SEM, *p < 0.05. B, Compartmented cultures were stimulated at cell bodies (CB) or distal axons (DA) for 8 h with neurotrophins. Cell body lysates were analyzed by Western blotting for Bcl-w (recombinant-Bclw was the positive control). DA stimulation leads to an increase in Bcl-w protein levels (32 ± 5%). C, The Trk kinase inhibitor K252a or vehicle control was applied to cell bodies and distal axons at 200 nm, a concentration that inhibits phosphorylation of Trk receptors in these neurons. Neurons in compartmented cultures were globally treated or not with K252a, and DA were stimulated with neurotrophins. K252a prevents induction of c-fos, bcl-w and mef2d by target-derived neurotrophins. D, The Erk kinase inhibitor UO126 or vehicle control was applied to cell bodies and distal axons at 50 nm, a concentration that inhibits phosphorylation of ERK kinases. Neurons in compartmented cultures were treated or not with UO126. UO126 prevents bcl-w, mef2d, and c-fos induction by target-derived neurotrophins. Neurons were infected with DN–MEK5 adenovirus for 3 d, then distal axons were stimulated with neurotrophins. DN–MEK5 expression inhibits bcl-w and mef2d mRNA induction but not c-fos induction by target-derived neurotrophins. E, Lentivirus expressing mef2d RNAi (M2) inhibits induction of bcl-w and mef2d, but not induction of c-fos, in response to target-derived neurotrophins (C = control lentivirus). All data show means ± SEM, *p < 0.05. F, Bcl-w and mef2d mRNA expression is specifically induced by distal axon stimulation and not by global stimulation (cultures maintained for 5–7 d).

Figure 5.

Bcl-w transcription is ERK5 and MEF2-dependent. A, Schematic of bcl-w 5′ region. Sequence and locations of consensus MEF2-binding sites are indicated. The luciferase construct consisted of the 2.5 kB promoter region, followed by the necleotides ATG and luciferase reporter gene. B, Luciferase reporter assay using bcl-w promoter-Luc cotransfected with pTK–Renilla–Luc for normalization. Two days after transfection by nucleofection, neurons were stimulated with neurotrophins for 2 h. Bcl-w-luciferase expression is neurotrophin dependent. C, D, Bcl-w promoter is ERK5 and MEF2 dependent. Middle and right luciferase reporter assay using bcl-w promoter-Luc, 3xMRE-Luc, or basic Luce cotransfected into COS cells with Renilla luciferase and the indicated expression plasmid(s). Three days after transfection, luciferase activity was measured. Data are from three independent experiments, each of which was conducted in triplicate, and show means ± SEM, *p < 0.05.

Figure 6.

Bcl-w and other retrograde response genes are important for the survival of DRG sensory neurons. A, Representative images of DAPI staining in dorsal root ganglia from cervical and lumbar regions of the spinal cord in bcl-2+/+ and bcl-w−/− mice. Arrows indicate apoptotic cells. Scale bar, 10 μm. B, In vivo analysis of apoptosis. Bcl-w−/− P0 mice show an increase in cell death compared with WT littermates, in both cervical and lumbar DRGs. The percentage of cells in the DRG with condensed nuclei when visualized by DAPI staining are shown. Three animals of each genotype were used, and 3–6 dorsal root ganglia from each lumbar and cervical region were counted. All data show means ± SEM, *p < 0.0001. C, Survival of bclw−/− sensory neurons in compartmented cultures stimulated with neurotrophin applied to the distal axons (DA) or the cell bodies (CB). DRG neurons were dissected at E14 and seeded in compartmented cultures. Bcl-w−/− neurons are more prone to apoptosis in serum free-media compared with bcl-w+/+ and +/− under all conditions tested, *p < 0.05. D, Retrograde response genes: DRG neurons in compartmented cultures were stimulated with NGF and BDNF or vehicle applied either to DA or the cell bodies (CB), for 2 h. RNA was prepared and expression of decorin, alsin, enpeptidase, mef2d, HSF-1, IGF-1, bcl-w, and c-fos were assessed by quantitative RT-PCR and normalized to gapdh in the same sample. Fold induction was measured by comparing normalized level of expression in neurotrophin-treated/vehicle-treated cells for four experiments each involving five cultures for each condition (DA shown first in black). Statistical analysis by z-test, **p ≤ 0.05, *p ≤ 0.10 for a difference from one.

Figure 7.

Target-derived neurotrophins activate an ERK5/MEF2 transcriptional program to promote survival. A, Neurotrophin binding and Trk activation results in endocytosis of the activated receptor complex. B, The signaling endosome, including downstream messengers, is retrogradely transported by microtubule-dependent dynein motors. C, On reaching the cell body, phosphorylated ERK5 translocates to the nucleus. D, MEF2D is phosphorylated and activated in response to a retrograde ERK5 signal. E, Activated MEF2D initiates transcription of bcl-w, mef2d, and other retrograde response genes (RRGs) to promote survival mediated by target-derived neurotrophins. F, In the case of cell body neurotrophin stimulation, both ERK5 and ERK1/2 are activated; however, expression of bcl-w, mef2d, and other retrograde response genes are not induced. One possible mechanism is through suppression of ERK5 by the ERK1/2 pathway.