Glial cell line-derived neurotrophic factor (GDNF) induces neuritogenesis in the cochlear spiral ganglion via neural cell adhesion molecule (NCAM)

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Abstract

Glial cell line-derived neurotrophic factor (GDNF) increases survival and neurite extension of spiral ganglion neurons (SGNs), the primary neurons of the auditory system, via yet unknown signaling mechanisms. In other cell types, signaling is achieved by the GPI-linked GDNF family receptor α1 (GFRα1) via recruitment of transmembrane receptors: Ret (re-arranged during transformation) and/or NCAM (neural cell adhesion molecule). Here we show that GDNF enhances neuritogenesis in organotypic cultures of spiral ganglia from 5-day-old rats and mice. Addition of GFRα1-Fc increases this effect. GDNF/GFRα1-Fc stimulation activates intracellular PI3K/Akt and MEK/Erk signaling cascades as detected by Western blot analysis of cultures prepared from rats at postnatal days 5 (P5, before the onset of hearing) and 20 (P20, after the onset of hearing). Both cascades mediate GDNF stimulation of neuritogenesis, since application of the Akt inhibitor Wortmannin or the Erk inhibitor U0126 abolished GDNF/GFRα1-Fc stimulated neuritogenesis in P5 rats. Since cultures of P5 NCAM-deficient mice failed to respond by neuritogenesis to GDNF/GFRα1-Fc, we conclude that NCAM serves as a receptor for GDNF signaling responsible for neuritogenesis in early postnatal spiral ganglion.

Introduction

The major reason for severe hearing impairment is the loss of inner and outer hair cells (HCs), the sensory cells of the inner ear. This can be due to many causes, including aging, noise exposure and ototoxic drugs (Lefebvre et al., 2002). HC loss is often followed by degeneration of primary cochlear afferent neurons (Stamataki et al., 2006), the spiral ganglion neurons (SGNs), which relay auditory information to the brain. Furthermore, SGNs may also degenerate without HC loss (Starr et al., 2003). In cases of near or complete HC loss resulting in deafness, central auditory function, i.e. hearing, can be restored via electrical stimulation of surviving SGNs with a cochlear implant in humans. However, cochlear implantation, and thus electrically evoked auditory nerve activity, did not enhance SGN survival in pharmacologically deafened or congenitally deaf animal models (e.g. Chen et al., 2010). Thus, enhancement of SGN survival, initiation of dendritic outgrowth from these neurons and maintenance of their intrinsic electrophysiological features (Davis and Liu, 2011) are desirable to further improve the performance of this prosthesis.

Glial cell line-derived neurotrophic factor (GDNF), first identified as a trophic factor for embryonic midbrain dopaminergic neurons (Lin et al., 1993), has been found to be a potent SGN survival factor in vitro and in vivo (e.g. Yagi et al., 2000, Ylikoski et al., 1998). For intracellular signaling, GDNF first binds to glycosylphosphatidyl inositol (GPI) anchored GDNF-family receptor α1 (GFRα1), but, since GFRα1 lacks an intracellular signaling domain, it must recruit a transmembrane receptor to induce intracellular signaling. Ret (rearranged during transformation) and the 140 kDa isoform of NCAM (neural cell adhesion molecule) have each been identified as receptors that can bind to the GDNF/GFRα1 complex and induce subsequent intracellular signaling, primarily via the PI3K/Akt and/or MEK/Erk MAPK pathways (Airaksinen et al., 1999, Jing et al., 1996, Paratcha et al., 2001).

GDNF, GFRα1, Ret and NCAM have been detected in SGNs as well as in the cochlear sensory epithelium (Ohgami et al., 2010, Stöver et al., 2000, Whitlon and Rutishauser, 1990, Ylikoski et al., 1998). However, the signaling mechanisms of the GDNF/GFRα1 complex in SGNs have not been investigated, and it is not known whether the reported survival effects are mediated by NCAM, Ret of both molecules, downstream of the initial binding of GDNF to GFRα1. We, therefore, analyzed GDNF and GDNF/GFRα1 effects in vitro using neo- and perinatal rat and mouse SGNs in organotypic tissue culture, and examined the significance of intracellular signaling cascades by both Western blot analysis of the PI3K/Akt and MEK/Erk MAPK signaling pathways in combination with specific inhibitors of these pathways. We further evaluated the downstream effects of GDNF via GFRα1 in SGNs derived from the NCAM knock-out (KO) mouse. Since Ret KO mice die at birth, the importance of Ret as a receptor for postnatal GDNF signaling via GFRα1 could not be investigated.

Section snippets

GDNF, GFRα1, Ret and NCAM are expressed in the developing and adult SGNs of rats

To determine the components of the GDNF signaling complex and the downstream signal transduction pathways in the SG, we first measured the expression of components of cognate GDNF signaling complexes in SGNs. Therefore, reverse transcription PCR (RT-PCR) and Western blot analysis were performed to examine messenger RNA (mRNA) and protein levels in this ganglion. Cell lysates were prepared from rat SGNs at different time points during development before and after the onset of hearing (about

Discussion

In the present study, we have investigated the substrates of GDNF signaling in SGNs. We present evidence that GDNF utilizes the 140 kDa isoform of NCAM as a transmembrane receptor in SGNs after GFRα1 binding for mediating its neuritogenic effect in the neonatal cochlea. However, NCAM does not appear to influence SGN survival.

Conclusions

The present study demonstrates that developing SGNs in vitro respond to GDNF with an increase in the number of neurites, with addition of exogenous GFRα1 increasing this effect. Both PI3K/Akt and MEK/Erk signaling pathways mediate these in vitro effects. Moreover, our results suggest that considerable differences exist in GDNF-mediated activation patterns in SGNs before and after the onset of hearing. Thus, at early postnatal ages NCAM appears to be the dominant signaling receptor that partners

Animals

The local animal subject committees of the Veterans Affairs San Diego Health Care Systems and the University of Lübeck approved all procedures in accordance with the guidelines laid down by the National Institute of Health regarding the care and use of animals for experimental procedures (NIH Publication No. 85-23, revised 1996). Animals were deeply anesthetized with an intraperitoneal injection of rodent cocktail (ketamine 60 mg/kg, xylazine 6.4 mg/kg, acepromazine 1.2 mg/kg) before decapitation

Contributions

S.E., K.H.Y., E.C., A.O. and K.P. performed and analyzed rat P5 tissue culture the experiments; S.E. performed mouse tissue culture experiments; S.E., E.C. and G.L. performed Western blotting experiments; S.E. performed RT-PCR, H&E staining and immunohistochemistry. S.E. and G.L. performed confocal imaging as well as image analysis. A.L. analyzed H&E images. S.E. and A.F.R. designed the experiments and obtained funding. S.E., G.L., M.S. and A.F.R. interpreted data and wrote the article.

Acknowledgments

We thank Dr. Gary D. Housley (University of New South Wales, Sydney) for advice on neurite labeling in whole mount preparations and Brendan Brinkman (UCSD) for continuous technical support at UCSD's confocal microscopy core facility. We thank Dr. Constantini (Columbia University, New York) for generously providing the Ret KO mouse line. Achim Dahlmann (Zentrum fuer Molekulare Neurobiologie Hamburg) and Sylvia Grammerstorf-Rosche (University of Lübeck) helped with genotyping of the NCAM mutant

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