Elsevier

Brain Research

Volume 1430, 9 January 2012, Pages 25-34
Brain Research

Research Report
Ras/p38 and PI3K/Akt but not Mek/Erk signaling mediate BDNF-induced neurite formation on neonatal cochlear spiral ganglion explants

https://doi.org/10.1016/j.brainres.2011.10.054Get rights and content

Abstract

Neurotrophins participate in regulating the survival, differentiation, and target innervation of many neurons, mediated by high-affinity Trk and low-affinity p75 receptors. In the cochlea, spiral ganglion (SG) neuron survival is strongly dependent upon neurotrophic input, including brain-derived neurotrophic factor (BDNF), which increases the number of neurite outgrowth in neonatal rat SG in vitro. Less is known about signal transduction pathways linking the activation of neurotrophin receptors to SG neuron nuclei. In particular, the p38 and cJUN Kinase (JNK), mitogen-activated protein kinase (MAPK) pathways, which participate in JNK signaling in other neurons, have not been studied. We found that inhibition of Ras, p38, phosphatidyl inositol 3 kinase (PI3K) or Akt signaling reduced or eliminated BDNF mediated increase in number of neurite outgrowth, while inhibition of Mek/Erk had no influence. Inhibition of Rac/cdc42, which lies upstream of JNK, modestly enhanced BDNF induced formation of neurites. Western blotting implicated p38 and Akt signaling, but not Mek/Erk. The results suggest that the Ras/p38 and PI3K/Akt are the primary pathways by which BDNF promotes its effects. Activation of Rac/cdc42/JNK signaling by BDNF may reduce the formation of neurites. This is in contrast to our previous results on NT-3, in which Mek/Erk signaling was the primary mediator of SG neurite outgrowth in vitro. Our data on BDNF agree with prior results from others that have implicated PI3K/Akt involvement in mediating the effects of BDNF on SG neurons in vitro, including neuronal survival and neurite extension. However, the identification of p38 and JNK involvement is entirely novel. The results suggest that neurotrophins can exert opposing effects on SG neurons, the balance of competing signals influencing the generation of neurites. This competition could provide a potential mechanism for the control of neurite number during development.

Highlights

► BDNF increases neonatal spiral ganglion survival and neurite number but not length in vitro. ► Inhibition of p38, Ras, PI3 kinase and Akt II signal transduction reduced BDNF-induced responses, while inhibition of Mek/Erk had no effect. ► Western blotting revealed specific activation of Akt and p38, but not Erk cell signaling in spiral ganglion neurons by BDNF. ► Neurotrophins can exert opposing effects on spiral ganglion neurons, competing signals influence cell survival and the generation of neurites.

Introduction

Neurotrophins play a critical role in neural development, regulating differentiation, neurite extension, target innervation and survival (Bibel and Barde, 2000). Brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are well known to influence neurons in the inner ear.

In particular, mice deficient in BDNF exhibit reduced cochlear neuronal populations, especially in the apical turn (Bianchi et al., 1996, Ernfors et al., 1994, Ernfors et al., 1995, Farinas et al., 2001, Fritzsch et al., 1997a, Fritzsch et al., 1997b). We, and others, have noted a dramatic effect of BDNF on developing spiral ganglion (SG) neurons in culture. BDNF treatment enhances survival of dissociated SG neurons (Malgrange et al., 1996), dramatically increases neurite number on SG explants (Pirvola et al., 1994) and promotes SG neurons survival in vivo (Leake et al., 2011).

Recently, Leake et al. (2011) demonstrated in neonatally deafened kittens and Landry et al. (2011) in adult deafened guinea pigs that chronic BNDF delivery from a miniosmotic pump improved electrically evoked auditory brainstem response thresholds. The authors therefore concluded that BDNF may have potential therapeutic value for the use with cochlear implants in the future. Furthermore, increasing reports are available on the potential therapeutic role of BDNF in a range of central nervous system (CNS) disorders such as amyotrophic lateral sclerosis, Parkinson's disease, peripheral neuropathy, Alzheimer's disease, Huntington's disease and stroke (reviewed by Nagahara and Tuszynski, 2011).

Neurotrophins signal primarily via high-affinity tyrosine kinase receptors in the cochlea, TrkB and TrkC (Pirvola et al., 1994), with some contribution from the low-affinity p75 receptor (Schecterson and Bothwell, 1994). BDNF signaling is mainly mediated via TrkB receptors and TrkB and p75 receptors are expressed by SG neurons throughout the inner ear (Knipper et al., 1996, Pirvola et al., 1994, Sano et al., 2001). Mice null for TrkB are reported to lose 15–20% of SG neurons (Fritzsch et al., 1997a, Fritzsch et al., 1997b). BDNF increases neurite number on SG explants in vitro throughout the entire length of the cochlea with no difference in the responses from different cochlear turns (our own unpublished data).

We previously found that Ras or Mek/Erk inhibition blocked NT-3 effects on SG neurites, while p38 inhibition had no effect (Aletsee et al., 2001). Mice with mutations in the docking site for the Shc adaptor protein on the TrkB receptor, which would be expected to reduce both Ras/MAPK and phosphatidyl inositol 3 kinase (PI3K) signaling, showed modest reduction in SG neuron survival (Postigo et al., 2002).

To explore BDNF signal transduction in SG neurons, SG explants were treated with BDNF in the presence of specific inhibitors of intracellular signaling pathways involved in TrkB signaling in the inner ear and other neuronal systems, and activation of signaling proteins was assessed by Western blotting.

Section snippets

BDNF increases SG neurite number but not length

Consistent with previous studies (Hartnick et al., 1996, Hegarty et al., 1997), treatment of neonatal SG explants with BDNF resulted in a significant increase (p < 0.05) in the number of SG neurites present on each explant (Fig. 1, Fig. 2). In contrast, and also consistent with prior results (Malgrange et al., 1996), there was no effect of BDNF treatment on the length of SG neurites (Fig. 1, Fig. 3).

Inhibitors of several signal transduction pathways alter BDNF-induced increases in SG neurite number

The influence of signaling inhibitors on the BDNF-induced increase in neurites on SG explants is

Discussion

In the current study, we show that Ras/P38 and PI3K/Akt but not Mek/Erk signaling mediate BDNF-induced neurite formation on neonatal cochlear SG explants. In order to assess the signaling pathways mentioned above, we first evaluated the effects of BDNF alone on SG neurites in vitro. Then, SG explants were treated with BDNF in the presence of specific inhibitors of the intracellular signaling pathways involved downstream from TrkB signaling. Finally, we confirmed activation of signaling proteins

Culture of spiral ganglion neurons

Surgical procedures were approved by the animal subject committee of the San Diego VA Medical Center in accordance with the guidelines laid down by NIH regarding the care and use of animals for experimental procedures. Three to five day old Sprague–Dawley rat pups (P3–P5) were decapitated and the skulls were opened midsagitally under sterile conditions. The membranous labyrinth was exposed by peeling off the cartilaginous cochlear capsule under a dissecting microscope. The stria vascularis and

Acknowledgments

Supported by the Research Service of the VA (AFR), NIH/NIDCD grant R01-DC000139 (AFR) and by Swiss National Science Foundation grant PBBSP3-13097 (YB).

References (48)

  • U. Pirvola et al.

    Coordinated expression and function of neurotrophins and their receptors in the rat inner ear during target innervation

    Hear. Res.

    (1994)
  • L. Rüttiger et al.

    BDNF mRNA expression and protein localization are changed in the age-related hearing loss

    Neurobiol. Aging

    (2007)
  • L.P. Rybak et al.

    Development of endocochlear potential and compound action potential in the rat

    Hear. Res.

    (1992)
  • L.C. Schecterson et al.

    Neurotrophin and neurotrophin receptor mRNA expression in developing inner ear

    Hear. Res.

    (1994)
  • E.F. Wheeler et al.

    Expression of BDNF and NT-3 mRNA in hair cells of the organ of Corti: quantitative analysis in developing rats

    Hear. Res.

    (1994)
  • J. Ylikoski et al.

    Expression patterns of neurotrophin and their receptor mRNAs in the rat inner ear

    Hear. Res.

    (1993)
  • C.L. Adamson et al.

    Opposite actions of brain-derived neurotrophic factor and neurotrophin-3 on firing features and ion channel composition of murine spiral ganglion neurons

    J. Neurosci.

    (2002)
  • K. Agerman et al.

    BDNF gene replacement reveals multiple mechanisms for establishing neurotrophin specificity during sensory nervous system development

    Development

    (2003)
  • C. Aletsee et al.

    Ras/MEK but not p38 signaling mediates neurite extension from spiral ganglion neurons

    JARO

    (2001)
  • L.M. Bianchi et al.

    Degeneration of vestibular neurons in late embryogenesis of both heterozygous and homozygous BDNF null mutant mice

    Development

    (1996)
  • M. Bibel et al.

    Neurotrophins: key regulators of cell fate and cell shape in the vertebrate nervous system

    Genes Dev.

    (2000)
  • D.E. Bredensen et al.

    p75NTR and apoptosis: Trk-dependent and Trk-independent effects

    Trends Neurosci.

    (1997)
  • D. Brors et al.

    Clostridium difficile toxin B, an inhibitor of the small GTPases Rho, Rac, and Cdc42, influences spiral ganglion neurite outgrowth

    Acta Otolaryngol.

    (2003)
  • D. Brors et al.

    EphA4 provides repulsive signals to developing cochlear ganglion neurites mediated through ephrin-B2 and -B3

    J. Comp. Neurol.

    (2003)
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    Drs. Ryan and Brand contributed equally to the supervision of this work.

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