BDNF synthesis in spiral ganglion neurons is constitutive and CREB-dependent

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Abstract

Brain-derived neurotrophic factor (BDNF), which supports spiral ganglion neuron (SGN) survival in vivo and in vitro, is synthesized by SGNs. The BDNF gene generates multiple different transcripts, each from its own promoter region. Using reverse transcriptase-polymerase chain reaction (RT-PCR), we find that SGNs express only the downstream transcripts III and IV in vivo and in vitro. Using RT-PCR assays of BDNF transcripts and transfection of BDNF promoter–reporter constructs, we tested the hypothesis, originally derived from studies of cortical neurons, that depolarization induces BDNF expression via a signaling pathway that includes Ca2+/calmodulin-dependent kinases (CaMKs) and the transcription factor, Ca2+/cyclic AMP response element binding protein (CREB). In contrast, we found that in SGNs in vivo BDNF expression is constitutive and is not increased by electrical activation. Similarly, BDNF expression in vitro is not increased by stimuli that activate CREB, including depolarization, cAMP, or transfection of activated CaMK mutants. However, transfection of dominant-negative CREB mutants did abrogate gene expression driven by BDNF promoters III and IV, indicating that CREB is necessary for constitutive BDNF expression. Thus, BDNF synthesis within SGNs makes possible an autocrine or paracrine mechanism that can contribute to support SGN survival but SGNs are distinctive in that this mechanism is constitutive and not activity-regulated.

Introduction

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family, which also includes nerve growth factor, neurotrophin-3 (NT-3), and neurotrophin-4 (Chao, 1992). The neurotrophins signal through Trk family receptor protein-tyrosine kinases with BDNF signaling through TrkB. BDNF promotes survival of a wide variety of neurons, regulates axonal and dendritic growth, and has been implicated in synaptic plasticity in the mammalian central nervous system (CNS) (Lindholm et al., 1994, Thoenen, 1995, Lewin and Barde, 1996).

Spiral ganglion neurons (SGNs) of the mammalian cochlea express TrkB (Pirvola et al., 1994, Schecterson and Bothwell, 1994, Vazquez et al., 1994, Knipper et al., 1996) and so can respond to BDNF. The survival of cultured mammalian or avian SGNs can be supported by BDNF (Avila et al., 1993, Lefebvre et al., 1994, Zheng et al., 1995, Hegarty et al., 1997, Mou et al., 1997). SGNs die gradually in vivo following deafferentation due to loss of hair cells, their sole presynaptic input, with the time required varying, depending on the species, from 3 months (e.g., rat) to several years (e.g., cat) (Spoendlin, 1975, Webster and Webster, 1981, Koitchev et al., 1982, Bichler et al., 1983). Although BDNF is not produced in the hair cells, the survival of deafferented SGNs can be maintained in vivo by infusion of BDNF into the cochlea (Staecker et al., 1996, Miller et al., 1997) or infection of the cochlea with a herpesvirus BDNF expression vector (Geschwind et al., 1996). These data indicate that BDNF is sufficient to maintain SGN survival in vivo and in vitro. Mice with homozygous deletion of the BDNF gene have reduced numbers of neural crest- and placode-derived sensory neurons, including a nearly complete loss of vestibular neurons, but SGN number is only slightly reduced in these mice (Ernfors et al., 1994, Ernfors et al., 1995, Jones et al., 1994, Conover et al., 1995, Liu et al., 1995, Fritzsch et al., 1997a, Fritzsch et al., 1997b, Fariñas et al., 1998). Thus, at least during embryonic development, BDNF is not necessary for SGN survival. Whether BDNF is necessary for SGN survival in vivo in the postnatal or adult mammalian auditory system is not yet known.

A significant source of BDNF available to SGNs in vivo appears to be the SGNs themselves (Wiechers et al., 1999, Hansen et al., 2001), suggesting an autocrine or paracrine role for BDNF in SGN survival or physiology. Moreover, we have recently found that SGNs synthesize BDNF mRNA and protein in vitro and that blockade of Trk signaling in cultured SGNs reduces their survival (Hansen et al., 2001), indicating that an auto/paracrine neurotrophic mechanism contributes to SGN survival, at least in vitro.

Previous studies have demonstrated that neuronal BDNF expression can be dependent on depolarization (Zafra et al., 1990, Zafra et al., 1992, Ernfors et al., 1991, Isackson et al., 1991, Gall, 1992, Larmet et al., 1992, Timmusk et al., 1993, Ghosh et al., 1994, Robinson et al., 1996). This implies that autocrine support of neuronal survival is regulated by depolarization and, moreover, that depolarization, to promote neuronal survival, uses an autocrine (Ghosh et al., 1994) or paracrine (Robinson et al., 1996) mechanism.

The mechanism by which depolarization regulates BDNF transcription is apt to be complex simply because BDNF transcription is a complex affair. The BDNF gene possesses at least four 5′ non-coding exons, any single one of which can be alternatively spliced to a common fifth 3′-exon that contains the protein coding sequence (Timmusk et al., 1995, Timmusk et al., 1999). We have also found evidence for a fifth 5′-exon in this gene (Bishop et al., 1994). Thus, multiple alternative transcripts are possible, in each of which a different one of the 5′ non-coding exons is spliced to the coding sequence. (For each of these a pair of transcripts is produced due to use of alternative polyadenylation signals in the 3′ non-coding sequence but this has not been implicated in regulation of expression.) Because each of the multiple 5′-exons has its own distinct 5′-flanking regulatory region, each of the alternative transcripts is independently regulated (Timmusk et al., 1993) and BDNF expression in a cell is the sum of expression due to each individual promoter. Previous studies have focused on promoter III (Shieh et al., 1998, Tao et al., 1998) in the direct regulation of BDNF expression by depolarization. Further analysis of the signal transduction mechanism used to link depolarization to BDNF gene regulation in neurons has implicated Ca2+ as a second messenger acting via Ca2+/calmodulin-dependent protein kinases (CaMKs) to phosphorylate and activate the transcription factor, Ca2+/cyclic AMP response element binding protein (CREB) (Shieh et al., 1998, Tao et al., 1998). However, different subsets of the promoters have been variously reported as being involved in regulation by depolarization in different cell types in different studies (Timmusk et al., 1993, Timmusk et al., 1995, Bishop et al., 1994, Lauterborn et al., 1996), making it difficult to generalize about BDNF regulation by depolarization in all neurons.

Because depolarization is a potent survival-promoting stimulus for SGNs (Hegarty et al., 1997) and because of the potential significance of BDNF as an autocrine trophic factor for SGNs, we asked here whether BDNF gene expression is regulated by membrane electrical activity in SGNs in vivo and in vitro. We also assessed the activity of the BDNF promoters by transfection of BDNF promoter–reporter constructs into SGNs in vitro. We found that BDNF expression in SGNs is largely constitutive both in vitro and in vivo and identified the promoters responsible. Surprisingly, although CREB activity is necessary for constitutive BDNF promoter activity, agents that increase CREB activity do not cause elevation of BDNF expression above the constitutive level. Depolarization does not cause a sustained elevation of BDNF expression that could account for the survival-promoting effect of depolarization.

Section snippets

Cell culture and transfection

Culture of spiral ganglion neurons (SGNs) was as described previously (Hegarty et al., 1997). Briefly, spiral ganglia were dissected out from postnatal day 5 (P5) rats. For transfection, dissociated SGNs were plated onto eight-well chamber slides (Lab-Tek) in high glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with N2 (Life Technologies) and 30 mM K+ (30K).

Transfection was carried out about 10 h after the cells were plated using a calcium phosphate-based protocol modified from

BDNF expression level in SGNs in vitro is independent of depolarization

In cortical neurons, BDNF expression in vitro is induced by depolarization (Ghosh et al., 1994). To determine whether BDNF levels might respond to depolarization, we cultured spiral ganglion cells taken from P5 rats for 48 h under depolarizing (30K) or non-depolarizing (5.4 mM [K+]o, 5K) conditions. BDNF protein was then detected by immunofluorescence. As shown in Fig. 1B, BDNF immunoreactivity was detected in SGNs 48 h after culture in 30K. However, BDNF immunoreactivity was also detected in

The expression of BDNF in SGNs

We show here that SGNs from P5 rat pups express the peptide neurotrophic factor BDNF in vitro. This is consistent with the results of Wiechers et al. (1999) who have shown an upregulation of BDNF expression in the spiral ganglion in postnatal rats in vivo. Since SGNs themselves express the BDNF receptor TrkB (Mou et al., 1997), and can be supported by BDNF in vivo and in vitro (Avila et al., 1993, Lefebvre et al., 1994, Zheng et al., 1995, Geschwind et al., 1996, Staecker et al., 1996, Hegarty

Acknowledgements

Support for this study was from NIH Grant DC02961 (S.H.G.). M.R.H. was supported by NIH training Grant DC00040. These studies made use of facilities and services provided by the University of Iowa Diabetes and Endocrinology Research Core, funded by NIH Grant DK25295, and the Statistical Consulting Center. We thank Dr. Charles Miller (University of Iowa, Department of Speech Pathology) for advice and invaluable discussion, May Wu and Xiaobing Du for technical assistance, and members of the Green

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    Present address: Cell Signaling Technology, 166B Cummings Center, Beverly, MA 01915, USA.

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