- Top of page
- Materials and methods
- Supporting Information
We investigated effects of Neuregulin 1 (NRG1) on the expression of nicotinic acetylcholine receptor (nAChR) in major pelvic ganglion (MPG) from adult rat. MPG neurons were found to express transcripts for type I and III NRG1s as well as α and β-type epidermal growth factor (EGF)-like domains. Of the four ErbB receptor isoforms, ErbB1, ErbB2, and ErbB3 were expressed in MPG neurons. Treating MPG with NRG1β significantly increased the transcript and protein level of the nAChR α3 and β4 subunits. Consistent with these molecular data, nicotinic currents (IACh) were significantly up-regulated in NRG1β-treated sympathetic and parasympathetic MPG neurons. In contrast, the type III NRG1 and the α form of the NRG1 failed to alter the IACh. Inhibition of the ErbB2 tyrosine kinase completely abolished the effects of NRG1β on the IACh. Stimulation of the ErbB receptors by NRG1β activated the phosphatidylinositol-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK). Immunoblot analysis revealed that PI3K-mediated activation of Akt preceded Erk1/2 activation in NRG1β-treated MPG neurons. Furthermore, specific PI3K inhibitors abrogated the phosphorylation of Erk1/2, while inhibition of MEK did not prevent the phosphorylation of Akt. Taken together, these findings suggest that NRG1 up-regulates nAChR expression via the ErbB2/ErbB3-PI3K-MAPK signaling cascade and may be involved in maintaining the ACh-mediated synaptic transmission in adult autonomic ganglia.
Neuregulin 1 (NRG1) belongs to a family of growth and differentiation factors that contain an epidermal growth factor (EGF)-like domain, and activates the ErbB family of receptor tyrosine kinases (ErbB2, ErbB3, and ErbB4) and are crucial in the development and maintenance of the nervous system. Alternative splicing and use of cell-specific promoters generates multiple NRG1 isoforms that have different localization patterns and functions. These isoforms can be categorized into three groups (type I, II, and III) based on the NH2-terminal characteristics of their extracellular domain (Buonanno and Fischbach 2001; Falls 2003; Esper et al. 2006). NRG1 binds to either ErbB3 or ErbB4, which stimulates receptor dimerization forming either heterodimeric ErbB2/ErbB3 or homodimeric ErbB4/ErbB4 (Riese and Stern 1998). Receptor dimerization activates the intrinsic receptor tyrosine kinase of ErbB2 and ErbB4, which results in the phosphorylation of specific tyrosine residues within the cytoplasmic tail of the receptor. These events activate multiple downstream signaling pathways that produce various biological activities.
NRG1 was originally discovered as a regulator of the expression of the nicotinic acetylcholine receptor (nAChR) in skeletal muscles during development. Among the three isoforms, type I NRG1 (originally named ARIA, acetylcholine receptor-inducing activity) has been implicated in neuromuscular junction (NMJ) formation by stimulating muscular nAChR expression during development (Martinou et al. 1991; Corfas et al. 1993; Falls et al. 1993). The signaling events that mediate the actions of ARIA in muscles involve either the activation of mitogen-activated protein kinase (MAPK), phosphatidylinositol-3-kinase (PI3K), or cyclin-dependent kinase (CDK) pathway (Si et al. 1996; Tansey et al. 1996; Altiok et al. 1997; Si and Mei 1999; Lu et al. 2005).
NRG1 also plays critical roles in the development of autonomic ganglia. For example, NRG1 promotes cell motility as neural crest cells migrate to form sympathetic ganglia (Young et al. 2004; Britsch et al. 1998). As in the skeletal muscles, NRG1 has also been shown to aid in the formation of nicotinic synapses by increasing the expression of nAChR subunits in embryonic chick sympathetic neurons (Yang et al. 1998). Currently, however, it is unknown whether NRG1 regulates the expression of nAChR in autonomic neurons during adulthood. Furthermore, the functional role of NRG1 in parasympathetic neurons has not been reported. We subsequently raised the following two questions: (i) Are NRG1 and its receptors still expressed in autonomic ganglion neurons during mammalian adulthood? and (ii) Is NRG1 required for the regulation of nAChR expression in adult autonomic neurons regardless of cell types (i.e., sympathetic and parasympathetic)? To address these questions, MPG was used because they contain sympathetic and parasympathetic neurons in one ganglion capsule (Dail et al. 1975) and have been well studied (Zhu et al. 1995; Lee et al. 2002; Park et al. 2006a, b; Won et al. 2006). Finally, we investigated the signaling pathways that underlie NRG1-induced expression of nAChR in adult MPG neurons.
- Top of page
- Materials and methods
- Supporting Information
Autonomic ganglion neurons express nAChR α3, α4, α5, α7, β2, and β4 subunits (Mandelzys et al. 1994; Poth et al. 1997; Park et al. 2006b). Among the conceivable subunit combinations comprising the heteropentameric nAChRs, the α3β4* is considered to be the primary combination in autonomic ganglia including the superior cervical ganglion (SCG) and MPG (Covernton et al. 1994; Rust et al. 1994; Park et al. 2006b; Albuquerque et al. 2009). Currently, however, there is little published data on how the nAChR α3β4* subtype is regulated in adult autonomic neurons. In this study, we report for the first time that NRG1 up-regulates the expression of the nAChR α3 and β4 subunits in both sympathetic and parasympathetic MPG neurons from adult rats.
MPG was found to express transcripts encoding type I and type III NRG1. In comparison to the whole ganglion, dissociated MPG neurons express type III NRG1 at a much lower level. This finding is consistent with a previous study where expression of the type III NRG1 is shown to be significantly reduced in adult dorsal root ganglion neurons when compared with embryonic neurons (Syed and Kim 2010). Therefore, the primary sources of the transcript encoding the type III NRG1 may be the pre-synaptic terminal and/or non-neural cells (e.g. glial cells) in MPG. Previous studies indicate that pre-synaptic inputs are critical for controlling nAChR expression at the synapses of developing autonomic neurons (Rosenberg et al. 2002). In embryonic sympathetic neurons, the type III NRG1 functions as a pre-synaptic input-derived regulatory factor that increases nAChR expression (Yang et al. 1998). Of the two isoforms, however, only the type I NRG1 was found to increase nAChR expression in adult autonomic neurons. This finding supports the notion that regulation of functional nAChR expression in mature autonomic neurons is less dependent on pre-ganglionic interactions (Zhou et al. 1998). It is unclear, however, whether the type III NRG1 plays other functional roles in adult autonomic ganglia. Recent findings suggest that the type III NRG1 can participate in bidirectional juxtacrine signaling. For example, the type III NRG1 is involved in maintaining Schwann cells in the pre-synaptic axons of the somatic motor neurons innervating the diaphragm muscles (Wolpowitz et al. 2000). The type III NRG1 is also involved in the axonal targeting of extrasynaptic nAChR subunits such as α7 in sensory neurons (Hancock et al. 2008) and CNS neurons (Zhong et al. 2008). Thus, it would be interesting to determine if the type III NRG1–ErbB interaction results in backward signaling within the pre-synaptic region of autonomic neurons. Unlike the type III NRG1, which is bound to the pre-synaptic membrane, the type I NRG1 acts in a paracrine manner after proteolytic cleavage (Loeb and Fischbach 1995; Loeb et al. 1998). In a different series of experiments, we observed that cleaved type I NRG1 is released from MPG neurons by either the electrical stimulation of sympathetic or parasympathetic pre-ganglionic nerves (i.e., hypogastric nerves or pelvic nerves, respectively) or by high potassium-induced depolarization, which increases nAChR expression (Kim et al., paper in preparation). Taken together, the type I NRG1 appears to regulate nAChR expression in adult MPG neurons. Retrograde signals from target tissues are also essential for maintaining nAChR levels in CNS and PNS neurons (Rosenberg et al. 2002; Albuquerque et al. 2009). CNTF, a trophic factor expressed in muscle cells innervated by avian ciliary ganglion neurons, up-regulates the α3 nAChR protein level (Finn and Nishi 1996). NGF is a potent regulator of α3 and β4 transcription in PC12 cells (Rogers et al. 1992; Henderson et al. 1994; Hu et al. 1994). In this study, however, these target-derived neurotrophic factors (CNTF, NGF, and BDNF) did not alter the nAChR expression level in the MPG neurons from adult rats. A previous study showed that neurotrophic factors regulate the NRG1 expression in embryonic ventral spinal cord neurons (Loeb and Fischbach 1997). Therefore, it is possible that neurotrophic factors indirectly participate in regulating the nAChR expression level by controlling the expression level of NRG1 in autonomic neurons. In MPG neurons, we detected both NRG1α and NRG1β transcripts, which are splice variants of the EGF-like domain. NRG1β has a significantly greater affinity for receptors and consequently is more biologically active than the α isoform (Raabe et al. 1996; Jones et al. 1999). Consistent with these findings, NRG1β, but not NRG1α, was effective in up-regulating the nAChR current in sympathetic and parasympathetic MPG neurons.
MPG neurons express the ErbB1 (EGFR), ErbB2, and ErbB3 receptors, but not the ErbB4 receptor. This expression pattern for the ErbB receptors was commonly found in other types of autonomic neurons (see 'Results'). Among the receptors expressed in MPG neurons, only ErbB3 is known to bind to NRG1 (Riese and Stern 1998). Several studies have shown that ErbB3 can form a heterodimer with either EGFR or ErbB2 (an orphan receptor), when it binds NRG1 (Riese and Stern 1998). In our study, a selective ErbB2 tyrosine kinase inhibitor (AG825) completely blocked the NRG1-induced up-regulation of nAChR expression. A previous study has shown that activation of the EGFR by EGF is capable of transmodulating ErbB2 (Riese and Stern 1998). However, EGF failed to alter nAChR expression in our experiments, which suggests that the EGFR or the EGFR/ErbB2 complex is not involved in the NRG1-induced up-regulation of nAChR expression in MPG neurons. Overall, we concluded that ErbB2 is a dimerization partner of ErbB3 that mediates NRG1 signaling in the MPG neurons of adult rats.
Even though the effects of NRG1 on nAChR expression have been studied in different types of neurons (Yang et al. 1998; Liu et al. 2001; Hancock et al. 2008; Zhong et al. 2008), the signaling mechanisms underlying the function of NRG1 have not been extensively investigated. The activation of ErbB2/ErbB3 by NRG1 is capable of directly or indirectly stimulating multiple enzymes (PI3K, MAPK, Phospholipase C-γ, cyclin-dependent kinases, etc.) to mediate a variety of biological responses (Citri et al. 2003). Of the EGF family of receptors, only ErbB3 has the ability to couple to PI3K (Fedi et al. 1994; Soltoff et al. 1994). In our experiments, treating MPG neurons with NRG1 resulted in the activation of the PI3K pathway, which supports the involvement of ErbB3 in NRG1-induced up-regulation of nAChR expression. The involvement of the PI3K pathway in stimulating or inhibiting nAChR expression has been reported in the Sol 8 muscle cell line and chick embryo myoblasts, respectively (Tansey et al. 1996; Altiok et al. 1997). In MPG neurons, the PI3K pathway is found to be stimulatory. In addition to the PI3K pathway, activation of ErbB2/ErbB3 by NRG1 also activates the MEK/MAPK (Erk1/2) pathway, which mediates the up-regulation of nAChR expression in MPG neurons. The roles of the PI3K and MEK/MAPK pathways in the up-regulation of nAChR expression appear to be similar to their role in muscle cells (Tansey et al. 1996; Altiok et al. 1997). However, our results show that activation of both PI3K and MAPK is necessary for increasing nAChR expression in MPG neurons, which suggest that there is crosstalk between two pathways. The NRG1 signaling in MPG neurons is quite different from that in muscle cells (Tansey et al. 1996) and from NGF signaling in the same neurons (see 'Results') in which the enzymes are activated in parallel. As depicted in Fig. 10, PI3K and MAPK are activated sequentially when ErbB receptors are stimulated by NRG1. In this study, several lines of evidence support that PI3K operates upstream of MEK–MAPK. First, the peak phosphorylation level of Akt preceded that of Erk1/2. Second, specific PI3K inhibitors (LY294002 and wortmannin) prevented the phosphorylation of Erk1/2. Third, the inhibition of MEK using U0126 failed to inhibit the phosphorylation of Akt. Furthermore, the PI3K requirement for activating the MEK–MAPK pathway has been previously reported (King et al. 1997; Aksamitiene et al. 2011).
Figure 10. A proposed signaling cascade underlying the neuregulin 1 (NRG1)-induced up-regulation of nicotinic acetylcholine receptor (nAChR) expression in adult major pelvic ganglion (MPG) neurons. Binding of NRG1 to the ErbB3 receptor induces ErbB2/ErbB3 dimerization. Phosphorylation of the ErbB2/ErbB3 heterodimer recruits phosphatidylinositol-3-kinase (PI3K). Activation of PI3K induces phosphorylation of Akt, which is followed by MEK-Erk1/2 signaling to up-regulate expression of the nAChR α3 and β4 subunits. It is unknown how the PI3K-Akt pathway stimulates the MEK-Erk1/2 pathway. However, the crosstalk between these pathways may be mediated by Akt-activated PAK1 (not shown). Tyrosine kinase inhibitors (AG825 and genistein), PI3K inhibitors (LY294002), and an MEK inhibitor (U0126) blocked the NRG1-induced up-regulation of nAChR expression.
Download figure to PowerPoint
The synaptic strength in the autonomic ganglion is determined by several factors, including the quantal content, the number of post-synaptic nAChRs, and the geometry of the neuronal cells (Vernino et al. 2009). Therefore, our findings indicate that NRG1, especially type I, may be critical for maintaining the synaptic strength at mature cholinergic synapses by regulating the expression of nAChR in autonomic neurons. Increasing evidence indicates that the down-regulation of nAChRs in autonomic neurons is associated with autonomic dysfunctions, such as neurogenic impotence (Huang et al. 2011). In this regard, NRG1 may be of therapeutic value.