Regulation of sympathetic neuron differentiation by endogenous nerve growth factor and neurotrophin-3

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Neuroscience Letters 431 (2008) 241–246

Regulation of sympathetic neuron differentiation by endogenous nerve growth factor and neurotrophin-3 Rosa Andres 1 , Luis A. Herraez-Baranda, Jane Thompson 2 , Sean Wyatt, Alun M. Davies ∗ Life Sciences Building, School of Biosciences, Museum Avenue, Cardiff CF10 3US, Wales, United Kingdom Received 4 October 2007; received in revised form 23 November 2007; accepted 28 November 2007

Abstract Nerve growth factor (NGF) and neurotrophin-3 (NT3) play distinctive roles in sympathetic axon growth and target field innervation and are required for sympathetic neuron survival in vivo. To ascertain if these neurotrophins selectively regulate the expression of genes that determine the functional characteristics of differentiated sympathetic neurons, we measured the mRNA levels for several such genes in the superior cervical ganglion of NGF−/− , NT3−/− and wild type mouse embryos at a stage before excessive neuronal loss occurs in the absence of these neurotrophins. Despite the extensively documented ability of NGF to regulate the noradrenergic phenotype of sympathetic neurons, we found that tyrosine hydroxylase (TH) and dopamine ␤ hydroxylase (D␤H) mRNA levels were normal in NGF−/− embryos, but significantly reduced in NT3−/− embryos. In contrast, the ␤2 nicotinic acetylcholine receptor and PACAP receptor 1 mRNA levels were normal in NT3−/− embryos, but significantly reduced in NGF−/− embryos. Studies of mice lacking neurotrophin receptors suggested that the effects of NGF on gene expression require TrkA whereas those of NT3 require TrkA and p75NTR . These findings demonstrate that endogenous NGF and NT3 have distinctive and separate effects on gene expression in early sympathetic neurons and that these selective effects on gene expression require a different combination of neurotrophin receptors. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: NGF; NT-3; Development; Sympathetic neurons; Tyrosine hydroxylase

Neurons of the superior cervical ganglion (SCG) are an advantageous model for studying neuronal differentiation [10]. These neurons receive cholinergic innervation from preganglionic neurons and use either noradrenaline or acetylcholine as their principal neurotransmitter. Noradrenergic characteristics, like the expression of TH and D␤H, appear early in proliferating sympathetic neuroblasts while cholinergic characteristics are acquired by a small proportion of neurons much later in development [6]. Although bone morphogenetic proteins play a key role in initiating noradrenergic differentiation [27], an extensive literature has implicated NGF in regulating the expression of TH and D␤H in developing and mature sympathetic neurons. NGF increases and anti-NGF decreases the activities of TH and D␤H in the SCG of neonatal rodents [14,31]. NGF maintains synthesis ∗

Corresponding author. Tel.: +44 29208 74303; fax: +44 29208 74116. E-mail address: [email protected] (A.M. Davies). 1 Current address: Institution for Research in Biomedicine, Josep Samitier 1-5, 08028 Barcelona, Spain. 2 Current address: Cyclacel Pharmaceuticals Inc., James Lindsay Place, Dundee DD1 5JJ, Scotland. 0304-3940/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2007.11.045

of TH and D␤H in adult rodent SCG neurons (reviewed in [32]) and prevents the decline in TH levels in these neurons following axotomy [7,16]. Transuterine injection of NGF into mouse embryos increases TH activity in SCG neurons whereas antiNGF has the opposite effect [17], and administration of NGF to chicken embryos increases TH mRNA levels in the sympathetic chain [12]. NGF promotes a dose-dependent increase in TH mRNA and protein in cultured newborn rat SCG neurons [13,22,25] and induces TH expression in PC12 cells [9,30]. In addition to studies implicating NGF in regulating the noradrenergic phenotype of sympathetic neurons, it is well established that NGF and NT3 are both needed for the survival of embryonic sympathetic neurons in vivo [3,8,37]. NT3 promotes the growth of sympathetic axons along the vasculature to reach their final targets, whereas NGF is crucial for final target innervation and retrograde survival signalling [11,24]. NGF signals via the TrkA receptor tyrosine kinase, NT3 is able to signal via TrkA, TrkB or TrkC, and both neurotrophins bind to the common neurotrophin receptor p75NTR [15]. The expression of many of genes that are responsible for the characteristic functional properties of sympathetic neurons

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begins early in development before they become dependent on endogenous NGF and NT3 for survival. Because both TrkA and p75NTR are expressed during this early stage of development [33,35–37], we investigated whether mice lacking NGF [3], NT3 [5], TrkA [29] or p75NTR [21] have detectable deficits in gene expression. We selected several genes whose transcripts can be reliably quantified in SCG dissected from E15 embryos before significant cell death occurs in the absence of NGF and NT3 [8,37]. We find that endogenous NGF and NT3 have distinctive and unexpected roles in regulating the expression of genes that govern the physiological properties of sympathetic neurons. A highly sensitive competitive RT/PCR technique was used to quantify the levels of TH, D␤H, ␣7AchR, ␤2AchR, PAC1 and GAPDH mRNAs [34]. The RT and PCR reactions were calibrated by spiking the RT reactions with known amounts of cRNA competitors that had a 3 bp insert between the primer sites. The primers were: TH(5 ) 5 -CAGAGTTGGATAAGTGTCACC3 , TH(3 ) 5 -AAGGCAATCTCTGCAATCAGC-3 , D␤H (5 ) 5 -ACTCCAAAATGAAGCCTGAC-3 , D␤H(3 ) 5 -CGTGGGTTGTGGTAATGAA-3 , ␣7AchR(5 ) 5 -CCTTGATAGCACAGTACTTCG-3 , ␣7AchR(3 ) 5 -GTCATGGTGGTGATATCGCAG-3 , ␤2AchR(5 ) 5 -CGCTGCTGTTCAGCTTTGGC-3 and ␤2AchR(3 ) 5 -TTAGTAGCTGGACGGATCAGC-3 , PAC1(5 ) 5 -GGCTATTGCTATGCACTCTGACTG-3 , PAC1(3 ) 5 -TGTGATATTGTCCCACATGCC-3 , GAPDH(5 ) 5 -TCCAGTATGACTCCACTCAC-3 and GAPDH(3 ) 5 -TCCTGGAAGATGGTGATGG-3 . TH, D␤H, ␤2AchR and PAC1 were amplified by 34 cycles of 95 ◦ C for 60 s, 55 ◦ C for 60 s and 68 ◦ C for 60 s. ␣7AchR was amplified by 34 cycles of 95 ◦ C for 60 s, 53 ◦ C for 60 s and 68 ◦ C for 60 s. The PCR products were resolved on 8% non-denaturating polyacrylamide gels. The gel was stained for 15 min in the dark with 1× Syber Gold solution (a non-isotopic UV-sensitive dye that attaches to DNA). PCR products were viewed under UV illumination. A computer videoimaging system was used to measure the PCR band intensity. Dissociated cell cultures from E13 SCG were grown in polyornithine/laminin coated 35 mm culture dishes in defined medium as described [23]. Neuronal survival in non-transfected cultures was assessed by counting the number of neurons in a grid 3 and 24 h after plating and expressing the 24 h count as a percentage of the 3 h count. To provide a stringent test of the role of endogenous NGF in regulating noradrenergic differentiation in developing sympathetic neurons, we quantified the expression of TH and D␤H transcripts in the SCG of wild type and mice that are homozygous for a null mutation in the NGF gene [3]. Because NT3 has been reported to promote noradrenergic differentiation in long-term neural crest cell cultures [38] and promote cholinergic differentiation while reducing noradrenergic markers in embryonic sympathetic neuron cultures [2], we also studied the expression of TH and D␤H transcripts in the SCG of mice that are homozygous for a null mutation in the NT3 gene [5]. To avoid differences in the numbers of neurons in the SCG of wild type and mutant embryos complicating interpretation of gene expression data, we carried out the study at E15 before enhanced neuronal death is observed in the SCG of NGF- and NT3-deficient mice [8,37]. By this stage, SCG neurons have

begun to innervate their NGF-producing targets and express both TrkA and the common neurotrophin receptor p75NTR [35]. To our surprise, there was no significant difference in the levels of TH mRNA and D␤H mRNA in the SCG of E15 wild type and NGF−/− embryos. In contrast, the levels of both transcripts were significantly reduced in the SCG of NT-3−/− embryos and NGF−/− /NT-3−/− embryos (Fig. 1). These data demonstrate that NT3, rather then NGF, is required in vivo for the rise in TH and D␤H expression that occurs in the early SCG when many sympathetic axons are growing towards and starting to innervate their targets. Having uncovered a role for endogenous NT3 in regulating noradrenergic traits in developing sympathetic neurons, we expanded our study to include other phenotypic markers expressed by these neurons. We first used semi-quantitative RT/PCR to gauge the expression of several potential candidates to find ones expressed at sufficiently high levels at E15 to be reliably quantified. These included the ␣7 nicotinic acetylcholine receptor subunit (␣7-AchR), ␤2 nicotinic acetylcholine receptor subunit (␤2-AchR) and pituitary adenylate cyclase activating polypeptide receptor-1 (PAC1). The level of ␣7-AchR mRNA in the SCG of E15 NGF−/− embryos was not significantly different from wild type littermates, but was significantly reduced in both NT3−/− and NGF−/− /NT3−/− embryos (Fig. 1). This suggests that ␣7-AchR expression is regulated by endogenous NT3. In contrast, there were no significant differences in the levels of ␤2-AchR and PAC1 mRNAs between wild type and NT3−/− embryos, but these transcripts were significantly reduced in the SCG of both NGF−/− and NGF−/− /NT3−/− embryos. This implies that NGF and not NT3 regulates the expression of ␤2AchR and PAC1 in vivo. These results indicate that endogenous NGF and NT3 regulate the expression of different groups of genes in developing sympathetic neurons. Given the distinctive effects of endogenous NGF and NT3 on gene expression, we asked whether these neurotrophins exert their effects via different receptors. We focused on TrkA and p75NTR because transcripts for these receptors are expressed in the SCG as early as E13 [35], whereas transcripts for catalytic TrkC, an exclusive receptor for NT-3, are barely detectable in the developing SCG [37]. We studied the SCG of mice that have a targeted deletion in the TrkA gene resulting in the expression of a receptor lacking the catalytic intracellular kinase domain [29] and mice with a targeted deletion in the p75NTR gene resulting in the expression of a receptor lacking the extracellular ligandbinding domain [21]. There were significant reductions in the mRNA levels of all five differentiation markers in the SCG of E15 TrkA−/− embryos (Fig. 2) of similar magnitude to those seen in TH, D␤H and ␣7-AchR mRNAs in NT3−/− embryos and ␤2AchR and PAC1 mRNAs in NGF−/− embryos (i.e., between 30 and 60% lower than in wild type littermates). This suggests that TrkA is required for the effects of both NGF and NT3. In contrast, the deficiencies in p75NTR−/− embryos were very similar to those seen in NT3−/− embryos with significant reductions in the levels of TH, D␤H and ␣7-AchR mRNAs only. This suggests that the effects of NT3 on gene expression in developing sympathetic neurons require p75NTR in addition to TrkA.

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Fig. 1. Levels of TH, D␤H, ␤2AchR, ␣7AchR and PAC1 mRNAs relative to GAPDH mRNA measured by competitive RT/PCR in RNA extracted from the SCG of E15 wild type, NGF−/− , NT3−/− and NGF−/− ; NT3−/− littermates resulting from crossing NGF+/− ; NT3+/− mice. To facilitate comparison of the expression data for different transcripts, the data are normalized to 100 for the means of the wild type data for each transcript. The means and standard errors from 4 to 9 embryos of each genotype are shown for each data point (data from 28 embryos in total). * p < 0.05, ** p < 0.01 significant decrease relative to wild type.

NT3 is synthesized in the immediate vicinity of early sympathetic ganglia and by the blood vessels along which many sympathetic axons extend to reach their distal targets [8]. Because NT3 is required for proximal sympathetic axon extension in vivo [19], it could influence gene expression in immature sympathetic neurons in vivo either directly or indirectly by facilitating the growth of sympathetic axons to tissues that provide signals that influence gene expression. To distinguish between these possibilities we examined the effects of NT3 on nora-

drenergic gene expression in dissociated cultures established from E13 SCG. At this early stage of development, the SCG is comprised of proliferating neuroblasts and immature neurons, the majority of which survive up to 48 h in culture without added neurotrophic factors [23]. Because of the very small size of SCG at this stage of development and the practical constraints of obtaining sufficient material to set up these cultures, we restricted our analysis to TH mRNA, the most abundantly expressed noradrenergic transcript in sympathetic neuroblasts.

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Fig. 2. Levels of TH, D␤H, ␤2AchR, ␣7AchR and PAC1 mRNAs relative to GAPDH mRNA measured by competitive RT/PCR in RNA extracted from the SCG of E15 wild type and TrkA−/− littermates resulting from crossing TrkA+/− mice, and E15 wild type and p75NTR−/− littermates resulting from crossing p75NTR+/− mice. To facilitate comparison of the expression data for different transcripts, the data are normalized to 100 for the means of the wild type data for each transcript. The means and standard errors from 6 TrkA−/− and 6 TrkA+/+ embryos and from 4 p75NTR−/− and 4 p75NTR+/+ embryos are shown. * p < 0.05, ** p < 0.01 significant decrease relative to wild type.

The level of TH mRNA relative to GAPDH mRNA in dissociated E13 SCG cultures grown for 24 h in defined, serum-free medium with NT3 at either 2 or 50 ng/ml was approximately two-fold greater than in control cultures (Fig. 3). Cell counts just prior to RNA extraction revealed no significant difference in number between NT3-treated cultures and control cultures

(data not shown). These results clearly demonstrate that NT3 is capable of acting directly on sympathetic neuroblasts and immature sympathetic neurons to enhance the expression of a key determinant of the adrenergic phenotype. In addition to examining the ability of NT3 to up-regulate TH mRNA expression in early SCG cultures, we also inves-

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Fig. 3. Levels of TH mRNA relative to GAPDH mRNA in dissociated cultures established from E13 SCG incubated for 24 h in defined medium alone (Cont) and medium supplemented with either NT3 (2 or 50 ng/ml) or NGF (10 ng/ml). The data are normalized to 100 for the means of the control data (n = 4 per experimental condition). The levels of TH mRNA were significantly greater in neurotrophin-treated cultures compared with control cultures (p < 0.005). There were no significant differences in the levels of TH mRNA between NT3-treated and NGF-treated cultures.

tigated the effect of NGF on the level of TH mRNA in these cultures. We found that NGF promoted a similar increase in TH mRNA to that seen with NT3 (Fig. 3). Thus, although our analysis of TH mRNA expression in the SCG of NGFdeficient embryos has clearly demonstrated that NGF is not required for TH mRNA expression during early sympathetic neuron development in vivo, these in vitro results show that NGF is just as capable of up-regulating TH mRNA in sympathetic neuroblasts and immature sympathetic neurons as NT3. The reason why NT3 and not NGF performs this role in early sympathetic neuron development in vivo is most likely related to when and where these neurotrophins are synthesized in the embryo. Whereas NT3 is synthesized in the immediate vicinity of early sympathetic ganglia and along the routes taken by sympathetic axons to reach their distal targets [8], NGF is synthesized later in development in their distal targets [18]. Thus, sympathetic neuroblasts and immature sympathetic neurons are initially exposed to NT3, not NGF, early in their development, and this locally acting NT3 directly enhances noradrenergic differentiation. While our in vivo data clearly show that NGF is not required for early developmental increases in TH and D␤H, our data do not preclude a role for NGF in enhancing and/or maintaining the expression of these enzymes later in development when the neurons have become dependent on target-derived NGF for survival. Indeed, it has been extensively demonstrated that

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NGF treatment is capable of up-regulating the expression of TH and D␤H in embryonic and mature sympathetic neurons [7,12–14,16,17,22,25,31,32]. Although we too show that NGF is capable of up-regulating TH mRNA from the earliest stages of sympathetic neuron development in vitro, we conclusively demonstrate that NT3 rather then NGF performs this role in vivo. Previous work has shown that TrkA is necessary and sufficient for the survival effects of NGF and NT3 on sympathetic neurons [1,4,8,37]. Our finding that TrkA−/− embryos have quantitatively very similar deficits in the expression of all genes studied to those selectively affected in NGF−/− and NT3−/− embryos suggests that TrkA is required for the effects of both NGF and NT3 on gene expression in early sympathetic neurons. Our finding that NT3−/− and p75NTR−/− embryos have quantitatively very similar deficits affecting the same genes suggests that p75NTR is additionally required for the effects of NT3 on gene expression in these neurons. It is known that p75NTR affects TrkA signalling in distinctive ways depending on the neurotrophin ligand. For example, in cultured sympathetic neurons, binding of NGF to p75NTR shifts the NGF survival dose response to lower concentrations whereas binding of NT3 to p75NTR shifts the NT3 survival dose response to higher concentrations [20,28]. Thus, it is possible that p75NTR -dependent effects of NT3 on TrkA signalling might contribute to the selective influence of NT3 on gene expression in early sympathetic neurons. Alternatively, it is known that p75NTR can signal independently of Trk receptors, and lead to the activation of several transcription factors [26]. Thus, potentially distinctive effects of NT3 on p75NTR signalling may be responsible for its distinctive effects on gene expression. In conclusion, by careful quantification of gene transcripts in the early sympathetic ganglia of mice lacking neurotrophins and their receptors, we have discovered that NGF and NT3 selectively affect the expression of different genes governing the differentiated characteristics of developing sympathetic neurons and that these selective effects on gene expression require a different set of neurotrophin receptors. These results compliment recent elegant studies of the distinctive roles of NGF and NT3 in sympathetic axon growth and tissue innervation [11,24], and illustrate the multiple and distinctive ways in which these neurotrophins orchestrate key aspects of sympathetic neuron development. Acknowledgements We thank Patrik Ernfors for the NT3 mice, Heidi Phillips for the NGF mice, Rudiger Klein for the TrkA mice and KuoFen Lee for the p75NTR mice. This work was supported by the Wellcome Trust. References [1] D.J. Belliveau, I. Krivko, J. Kohn, C. Lachance, C. Pozniak, D. Rusakov, D. Kaplan, F.D. Miller, NGF and neurotrophin-3 both activate TrkA on sympathetic neurons but differentially regulate survival and neuritogenesis, J. Cell Biol. 136 (1997) 375–388.

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