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Presynaptic Type III Neuregulin1-ErbB signaling targets 7 nicotinic acetylcholine receptors to axons

¹ Integrated Program in Cellular, Molecular and Biophysical Studies, ² Department of Neurosciences, ³ Department of Pathology and Cell Biology, and ⁴ The Institute of Human Nutrition, Columbia University, New York, NY 10032

Correspondence to David A. Talmage: talmage{at}pharm.stonybrook.edu

Type III Neuregulin1 (Nrg1) isoforms are membrane-tethered proteins capable of participating in bidirectional juxtacrine signaling. Neuronal nicotinic acetylcholine receptors (nAChRs), which can modulate the release of a rich array of neurotransmitters, are differentially targeted to presynaptic sites. We demonstrate that Type III Nrg1 back signaling regulates the surface expression of 7 nAChRs along axons of sensory neurons. Stimulation of Type III Nrg1 back signaling induces an increase in axonal surface 7 nAChRs, which results from a redistribution of preexisting intracellular pools of 7 rather than from increased protein synthesis. We also demonstrate that Type III Nrg1 back signaling activates a phosphatidylinositol 3-kinase signaling pathway and that activation of this pathway is required for the insertion of preexisting 7 nAChRs into the axonal plasma membrane. These findings, in conjunction with prior results establishing that Type III Nrg1 back signaling controls gene transcription, demonstrate that Type III Nrg1 back signaling can regulate both short-and long-term changes in neuronal function.

D.A. Talmage's present address is Department of Pharmacological Science and Center for Brain and Spinal Cord Research, State University of New York at Stony Brook, Stony Brook, NY 11794.

Abbreviations used in this paper: AFI, average fluorescence intensity; Akt inh., Akt inhibitor; a.u., arbitrary units; CHX, cycloheximide; DRG, dorsal root ganglia; E, embryonic day; ECD, extracellular domain; nAChR, nicotinic acetylcholine receptor; NF, neurofilament; Nrg1, Neuregulin1; PAO, phenylarsine oxide; PIP₃, phosphatidylinositol 3,4,5 trisphosphate; PtdIns 3K, phosphatidylinositol 3-kinase; vGlut1, vesicular glutamate transporter 1; WM, wortmannin; WT, wild type.

© 2008 Hancock et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

	Introduction

Top Abstract Introduction Results Discussion Materials and methods References

 
The Neuregulin1 (Nrg1) gene encodes an extremely important and diverse family of proteins that signal by binding to the ErbB family of receptor tyrosine kinases. Nrg1-ErbB signaling regulates neural development, glial growth, myelination, and the maintenance of synaptic connections in both the peripheral and central nervous system (Michailov et al., 2004; Taveggia et al., 2005; Chen et al., 2006; Lopez-Bendito et al., 2006). Nrg1 activation of ErbB signaling regulates the levels of several ion channels, including subtypes of neuronal nicotinic acetylcholine receptors (nAChRs; Yang et al., 1998; Liu et al., 2001; Kawai et al., 2002; Chang and Fischbach, 2006), N-methyl-D-aspartic acid receptors (Ozaki et al., 1997; Gu et al., 2005), -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (Kwon et al., 2005; Li et al., 2007), and -amino butyric acid_A receptors (Rieff et al., 1999; Okada and Corfas, 2004). Nrg1 has been implicated as a schizophrenia susceptibility gene, and the potential relationship between Nrg1 and circuits affected in schizophrenia underscores the importance of determining the molecular mechanisms of action of Nrg1 signaling in the developing and adult nervous systems (Stefansson et al., 2002).

The Nrg1 gene encodes over a dozen different protein products. Multiple promoters and alternative splicing leads to the synthesis of three (and perhaps as many as six) classes of proteins differing in their amino-terminal domains, as well as in the EGF-like domain ( or β), and/or in the length of the C termini (Falls, 2003). These differences in primary sequence translate into subtle and dramatic differences in signaling strategies. Although Types I and II Nrg1 are either directly secreted or released after constitutive cleavage, Type III Nrg1 remains membrane tethered as a result of its unique N-terminal transmembrane domain (Wang et al., 2001; Taveggia et al., 2005). Thus, Types I and II Nrg1 can participate in paracrine signaling, whereas Type III Nrg1 predominantly signals in a juxtacrine manner. Additionally, it has been shown that Type III Nrg1 also functions as a receptor, in that Type III Nrg1–ErbB interaction results in signaling within the Type III Nrg1–expressing cell (Bao et al., 2003, 2004).

The integrity of a neuron–neuron synapse is determined by several molecular interactions between pre- and postsynaptic cells. Throughout the central and peripheral nervous system, nAChRs function as cation-selective ligand-gated ion channels and play important roles in synaptic signaling (Berg and Conroy, 2002). There is increasing evidence for the importance of nAChRs in the modulation of transmitter release from presynaptic terminals (McGehee et al., 1995; MacDermott et al., 1999), neuronal survival (Dajas-Bailador et al., 2000; Mechawar et al., 2004), and postsynaptic signaling (Zhang et al., 1996; Zhang and Berg, 2007). Individual nAChR channels have unique biological functions that are determined by the pentameric combination of 11 distinct subunits: 2–7, 9, 10, and β2–β4 (Rosenberg et al., 2002). Of particular interest are the 7* nAChRs (7-containing nAChRs) because of their high permeability to calcium and abundant expression in both mammalian and avian peripheral nervous systems (Roth and Berg, 2003; Fucile et al., 2005).

The Type III isoform of Nrg1 was identified as the nerve-derived factor responsible for increasing acetylcholine-evoked currents in neurons and was shown to regulate expression of neuronal nAChR subunit mRNAs, including that of 7 (Yang et al., 1998). More recent findings in our laboratory reveal a requirement for presynaptic Type III Nrg1 in potentiation of glutamatergic transmission by nicotine in a central sensory motor–gating circuit (Du, C., C. Zhong, M. Hancock, D.A. Talmage, and L.W. Role. 2004. Society for Neuroscience 34th Annual Meeting; unpublished data). These results inspired us to investigate the molecular mechanisms underlying presynaptic Type III Nrg1's cell-autonomous effect on functional 7* nAChRs along axonal projections.

In this paper, we show that presynaptic Type III Nrg1 is required for normal levels of presynaptically targeted 7* nAChRs in primary sensory neurons. Sensory neurons from Type III Nrg1 mutant mice exhibit deficits of the surface expression of 7* nAChRs. Acute stimulation of Type III Nrg1 back signaling increases the number of 7* nAChRs on sensory axons from wild-type (WT), but not from homozygous Type III Nrg1, mutant embryos. These increases in axonal surface 7* nAChRs result from redistribution of preexisting intracellular pools of 7 rather than from increased 7 protein synthesis. Increases in surface pools of 7* nAChR are accomplished, at least in part, by local activation of a phosphatidylinositol 3-kinase (PtdIns 3K) signaling pathway, which in turn stimulates the membrane insertion of intracellular 7* nAChRs.

	Results

Top Abstract Introduction Results Discussion Materials and methods References

 
Reduction of Type III Nrg1 expression results in decreased axonal 7 nAChR surface expression
Subsets of primary sensory neurons within the dorsal root ganglia (DRG) express Type III Nrg1 and 7 nAChR subunit mRNAs (Boyd et al., 1991; Wolpowitz et al., 2000; Genzen et al., 2001; Taveggia et al., 2005). In the following experiments, we examined the expression of 7* nAChRs and Type III Nrg1 on the surface of axons from sensory neurons of WT or Type III Nrg1^–/– mouse embryos. Embryonic neurons were used for these experiments because mutant mice lacking Type III Nrg1 die at birth (Wolpowitz et al., 2000).

To label surface 7* nAChRs, primary sensory neurons were labeled with BgTx-488 (Alexa 488–conjugated -bungarotoxin) before fixation and permeabilization (Fig. 1 A). -BgTx is an effective reagent for probing 7* nAChRs because of its high affinity for 7* nAChRs (Ravdin and Berg 1979). After fixation and permeabilization, we applied antibodies recognizing the cysteine-rich domain of Type III Nrg1 (red) or neurofilament (NF) proteins (blue; Fig. 1 A; Yang et al., 1998). In neurons from WT mice, axons were studded with distinct puncta of BgTx-488 and Type III Nrg1 along the same sensory axons. Pretreatment with 5 µM methyllycaconitine or 1 µM of nicotine before addition of BgTx-488 blocked surface labeling of 7* nAChRs, confirming the specificity of surface BgTx-488 labeling (Fig. 1 D). We repeated this analysis using sensory neurons isolated from Type III Nrg1^–/– embryos. Labeling with BgTx-488 identified discrete clusters of 7* nAChRs on the axonal surface of mutant neurons (Fig. 1 A). However, when we quantified the number of these BgTx-488 clusters we found that the mutants had 50% fewer clusters than WT neurons (WT, 6.47 ± 0.69 clusters/100 µm; Type III Nrg1^–/–, 3.22 ± 0.59 clusters/100 µm; Fig. 1 B). Additionally, we found that the mutants had a 35% reduction in mean cluster area relative to WT neurons (WT, 0.26 ± 0.02 µm²; Type III Nrg1^–/–, 0.17 ± 0.02 µm²; Fig. 1 C).

View larger version (40K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Decreased surface expression of 7* nAChRs along axons of Type III Nrg1–/– sensory neurons. Sensory ganglia were extirpated from embryonic day (E) 14.5 WT or Type III Nrg1–/– embryos and plated as explants. After 2 d in vitro, neurons were labeled for surface 7* nAChRs with BgTx-488 (green). Neurons were fixed, permeabilized, and stained for Type III Nrg1 (red) and NF protein (blue). (A) Representative micrographs of BgTx-488 clusters along NF-positive axons of WT and Type III Nrg1–/– sensory neurons at 2 d in vitro. Type III Nrg1 staining was detected along WT (a) but not Type III Nrg1–/– (d) axons. Fewer surface BgTx-488 clusters were detected along mutant axons (e) as compared with WT (b). Confocal images were acquired from a 100x oil objective. Bar, 5 µm. (B) Quantification of surface BgTx-488 clusters per 100 µm of axonal length revealed an 50% reduction of clusters along mutant axons. The graph shows means ± SEM. Data were pooled from three independent experiments. Statistical significance determined by ANOVA. *, P < 0.001 (Statview). (C) Quantification of the surface BgTx-488 cluster area. Loss of Type III Nrg1 expression resulted in an 35% reduction of BgTx-488 cluster area. Data pooled from three independent experiments were analyzed using nonparametric statistics and presented as box plots (see Materials and methods). Statistical significance was determined by the Kolmogorov-Smirnov Test. *, P < 0.0001 (Statview). (D) Pretreatment of sensory neurons with 1 µM of nicotine (Nic; b and e) or 5 µM methyllycaconitine (MLA; c and f) prevents surface BgTx-488 labeling along axons. Bar, 10 µm. (E) Immunoblot analysis of total 7 subunit protein levels in sensory neurons from E14.5 WT, Type III Nrg1+/–, and Type III Nrg1–/– embryos cultured for 2 d in vitro (a). MAPK1/2 probing in the bottom panel shows equal lysate loading. Immunoblots of total 7 subunit protein levels in brain extracts from WT or 7 nAChR–/– embryos are also shown (b). Glyceraldehyde 3-phosphate dehydrogenase probing in the bottom panel shows equal lysate loading (b). (F) In WT sensory neurons, nicotine application (1 µM for 1 min) resulted in an increased internal concentration of Ca2+ (indicated in pseudo color; described in Materials and methods). The white arrowhead highlights an axonal region affected by nicotine. Bar, 10 µm. (G) Changes in internal concentration of Ca2+ in response to application of nicotine in WT and mutant axons are plotted. Note that 100 nM BgTx completely eliminated the response to nicotine in WT axons and that sensory axons from Type III Nrg1–/– animals did not respond to nicotine. Data are from two independent experiments. The graph shows means ± SEM. Statistical significance was determined by ANOVA with post-hoc Fisher's PLSD test. *, P < 0.0001 (Statview).

To demonstrate that the BgTx-488 surface binding measurements reflected the presence of functional 7* nAChRs, we quantified internal concentrations of Ca²⁺ along sensory axons after a 1-min application of 1 µM of nicotine. In response to nicotine, we detected an increase in internal concentrations of Ca²⁺ within 1 min along axons of WT neurons (Fig. 1, F and G). When this experiment was repeated in the presence of 1 µM BgTx, we did not detect an increase in intracellular concentrations of Ca²⁺, indicating that the nicotine-induced changes were mediated by functional 7* nAChRs. Nicotine application did not increase internal Ca²⁺ concentrations in Type III Nrg1^–/– axons (Fig. 1 G), a result which is consistent with a significant decrease in surface 7* nAChRs in these neurons.

Type III Nrg1 back signaling increases surface expression of 7* nAChR clusters
Type III Nrg1 functions as a membrane-tethered bidirectional signaling molecule (Bao et al., 2003, 2004). To test the possibility that Type III Nrg1, acting as a receptor, contributes to the regulation of 7* nAChR levels on sensory axons, we treated WT or Type III Nrg1^–/– sensory neurons with 2 nM of either the extracellular domain (ECD) of ErbB2 (B2-ECD; control) or ErbB4 (B4-ECD) for 24 h (Fig. 2, A and B). B4-ECD, but not B2-ECD, binds with high affinity to the EGF-like domain of Nrg1 (Fig. S2 A, available at http://www.jcb.org/cgi/content/full/jcb.200710037/DC1; Fitzpatrick et al., 1998; Bao et al., 2003). We visualized 7* nAChRs present on the surface of sensory axons by labeling with BgTx-488 (green) before fixation (Fig. 2 A). Treating WT sensory neurons with B4-ECD for 24 h led to an increase in the number of BgTx-488 clusters on the axonal surface (from 6.47 ± 0.69 clusters/100 µm in control cultures to 10.38 ± 1.59 clusters/100 µm; Fig. 2 B). When we repeated the experiment using sensory neurons from Type III Nrg1^–/– embryos, there was no significant change in the number of BgTx-488 clusters on mutant axons (from 3.07 ± 0.72 clusters/100 µm in control cultures to 2.74 ± 1.14 clusters/100 µm in B4-ECD treated). This effect did not result from general changes in the levels of surface proteins, as we did not detect a change in the axonal surface levels of TrkA in response to a 24-h B4-ECD treatment (Fig. S1, E and F).

View larger version (44K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Type III Nrg1 back-signaling increases the surface expression of 7* nAChRs. E14.5 WT or Type III Nrg1–/– DRG explants were treated with B2-ECD (control) or B4-ECD for 24 h. Neurons were labeled for surface 7* nAChRs with BgTx-488 (green), fixed, permeabilized, and labeled for NF protein (blue). (A) Representative micrographs of axons from WT (a and b) or Type III Nrg1–/– (c and d) sensory neurons under control (a and c) versus B4-ECD (b and d) conditions. B4-ECD treatment increased the number of surface BgTx-488 clusters along NF-positive processes of WT neurons (b). Linescans of fluorescence intensity profile for BgTx-488 staining along representative axons (see Materials and methods). Bar, 5 µm. (B) Quantification of surface BgTx-488 clusters along NF-positive axons from WT versus Type III Nrg1–/– DRG explants treated with either B2-ECD (control) or B4-ECD for 24 h. In WT cultures, B4-ECD treatment induced an 1.6-fold increase in surface BgTx clusters along NF-positive axons compared with the control. There was no detectable change in BgTx clusters along axons of Type III Nrg1–/– neurons. The graph shows means ± SEM. Data were pooled from three independent experiments. Statistical significance was determined by ANOVA with post-hoc Fisher's PLSD test. *, P < 0.03; **, P < 0.001 (Statview). (C) After 2 d in vitro, dissociated sensory neurons from E11 chick embryos were treated with B2-ECD (control) or B4-ECD for 1, 2, 6, or 12 h and labeled as described in A. Axonal surface BgTx-488 clusters were quantified. Data were pooled from three independent experiments. The graph shows means ± SEM. Statistical significance was determined by ANOVA with post-hoc Fisher's PLSD test. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Statview). (D) Axonal-bound B4-ECD and BgTx-488 were detected in puncta along axons treated with B4-ECD (b, d, and e). Sensory neurons from E14.5 WT mouse embryos were cultured for 2 d in vitro and treated with B2-ECD (control) or B4-ECD for 1 h. Before fixation, surface 7* nAChRs and axonal-bound B2-ECD (control) or B4-ECD were labeled with BgTx-488 (green) and an antibody against the human Fc domain (anti-Fc; red), respectively. c and d and e are magnifications of the areas shown in dotted squares in a and b, respectively. Bar: (a and b) 5 µm; (c–e) 1 µm. (E) Sensory neurons from E11 chick embryos were treated with B2-ECD (control), B4-ECD, or soluble Nrg1β peptide (Nrg1-ECD) for 1 h. In parallel, neurons pretreated with an ErbB tyrosine kinase inhibitor (ErbB inh.) for 45 min were treated with B2-ECD, B4-ECD, or Nrg1-ECD for 1 h. Neurons were labeled as described in A, and surface BgTx-488 clusters along axons were quantified. Data were pooled from three independent experiments. The graph shows means ± SEM. Statistical significance was determined by ANOVA with post-hoc Fisher's PLSD test. *, P < 0.005; **, P < 0.01 (Statview).

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Type III Nrg1 back signaling increases the surface expression of 7* nAChRs in the absence of protein synthesis. Dissociated sensory neurons from E11 chick embryos were cultured for 2 d in vitro and treated with either B2-ECD (control) or B4-ECD for 1 or 24 h. (A) Quantification of surface or total pools of 7* nAChR by 125I-BgTx radiolabeling in sensory neurons treated with either B2-ECD (control) or B4-ECD for 24 h. In response to a 24-h B4-ECD treatment, we detected an 2.7-fold increase in surface 125I-BgTx binding compared with control conditions (B2-ECD [control], 1,339.15 ± 329.77 cpm; and B4-ECD, 3,562.81 ± 1,111.19 cpm). B4-ECD treatment did not induce a change in total 125I-BgTx binding as compared with the control (B2-ECD [control], 11,159.74 ± 1,059.79 cpm; and B4-ECD, 12,258.85 ± 580.11 cpm). The graph shows means ± SEM. Data were pooled from three independent experiments with greater than or equal to three wells per condition per experiment. Statistical significance was determined by ANOVA. *, P < 0.05 (Statview). (B) Immunoblot analysis of total 7 subunit protein in sensory neurons treated with B2-ECD (control) or B4-ECD treatment for 24 h. In response to B4-ECD treatment, we did not detect a difference in total 7 subunit protein. NFM probing in bottom panel shows equivalent lysate loading. (C) Sensory neurons were treated with B2-ECD (control) or B4-ECD for 1 h. In parallel, neurons pretreated with CHX for 45 min were treated with B2-ECD or B4-ECD for 1 h. Neurons were labeled with BgTx-488 (green), fixed, permeabilized, and colabeled for NF protein (blue). CHX treatment (c and d) did not affect either the basal number of BgTx-488 clusters on control neurons (c) or the response to B4-ECD (d). Linescans of fluorescence intensity profiles of BgTx-488 along representative axons (see Materials and methods) are shown. Bar, 5 µm. (D) Quantification of surface BgTx-488 clusters along NF-labeled axons. B4-ECD treatment induced an 1.9-fold increase in surface BgTx-488 clusters along axons, and B4-ECD treatment in the presence of CHX induced an 2.1-fold increase. Data were pooled from three independent experiments. The graph shows means ± SEM. Statistical significance was determined by ANOVA with post-hoc Fisher's PLSD test. *, P = 0.01; **, P < 0.0001 (Statview). (E) Quantification of surface BgTx-488 cluster area. B4-ECD treatment in the presence or absence of CHX induced an increase in BgTx-488 cluster area. Data pooled from three independent experiments were analyzed using nonparametric statistics and presented as box plots (see Materials and methods). Statistical significance was determined by the Kolmogorov-Smirnov Test. *, P 0.0001 (Statview).

Stimulation of Type III Nrg1 back signaling increases surface expression of 7* nAChRs in the absence of new protein synthesis
Stimulation of Type III Nrg1 back signaling elicits a transcriptional response mediated by the Nrg1 intracellular domain (Bao et al., 2003, 2004). To determine if Type III Nrg1–dependent regulation of axonal 7* nAChR levels resulted from Nrg1 intracellular domain–induced synthesis of 7 and/or changes in 7* nAChR surface expression along axons, we compared measures of total (surface and internal) neuronal ¹²⁵I-BgTx binding (Fig. 3 A) and total immunoreactive 7 nAChR protein (Fig. 3 B) in sensory neurons treated with B2-ECD (control) or B4-ECD for 24 h. We also asked whether B4-ECD treatment increased surface 7* nAChR levels in the presence of a protein synthesis inhibitor (Fig. 3, C–E).

Surface 7* nAChRs were quantified by ¹²⁵I-BgTx labeling in live in vitro preparations of chick sensory neurons and, in parallel, the total pool of 7* nAChRs was quantified by ¹²⁵I-BgTx binding after permeabilization with 0.5% saponin (Fig. 3 A). Under basal conditions, 12% of the total 7* nAChRs were on the surface, which is similar to prior results emphasizing the large internal pools of nAChRs in neurons (Cho et al., 2005). After a 24-h treatment with B4-ECD, we observed an 2.7-fold increase in surface ¹²⁵I-BgTx binding. In B4-ECD–stimulated neurons, 30% of the 7* nAChRs were at the surface, but the total pool of ¹²⁵I-BgTx–labeled sites was not changed. These results are consistent with Type III Nrg1 back signaling inducing a relocalization of 7* nAChRs from preexisting internal pools to clusters along the axonal surface. Results on increased surface versus total 7* nAChRs were confirmed by immunoblot assay (Fig. 3 B). These results indicate that in sensory neurons, Type III Nrg1, acting as a receptor, can increase axonal surface 7* nAChRs without significantly increasing the size of the total 7* nAChR pool.

To extend our examination of the idea that Type III Nrg1 back signaling stimulates insertion of preexisting 7* nAChRs into the axonal membrane, we repeated the B4-ECD stimulation of dispersed sensory neurons in the presence of the protein synthesis inhibitor cycloheximide (CHX; Fig. 3, C–E). Neurons were pretreated with 10 µg/ml CHX for 45 min and then stimulated for an additional hour with either B2-ECD (control) or B4-ECD. Neither the B4-ECD–induced increase in the number of surface BgTx-488 clusters (Fig. 3 D) nor the increase in cluster area (Fig. 3 E) were affected by the CHX pretreatment (CHX, 9.72 ± 1.65 clusters/100-µm axonal length; CHX + B4-ECD, 19.96 ± 2.83 clusters/100-µm axonal length; CHX, 0.23 ± 0.02 µm²; and CHX + B4-ECD, 0.32 ± 0.01 µm²). Thus, the increase in surface 7* nAChRs resulted from a redistribution of preexisting receptors rather than from new receptor synthesis.

Type III Nrg1 back signaling increases surface expression of 7* nAChRs in the absence of endocytosis
Prior studies have shown that endocytosis can alter the surface expression of neurotransmitter receptors (Man et al., 2007; Xia et al., 2007). Using a pharmacological inhibitor of endocytosis, phenylarsine oxide (PAO; Hertel et al., 1985), we investigated whether B4-ECD–mediated increase in the surface expression of 7* nAChR clusters was caused by alterations in receptor endocytosis. We applied PAO to sensory neurons for 45 min before B2-ECD (control) or B4-ECD treatment. Although PAO did not significantly alter the number of surface 7* nAChR clusters along axons under control conditions, it significantly increased the area of the clusters (from 0.2 ± 0.01 µm² under control conditions to 0.29 ± 0.03 µm² after PAO treatment; Fig. 4 C). After B4-ECD treatment for 1 h, we detected an increase in both the number and area of surface 7* nAChR clusters along axons when endocytosis was blocked (Fig. 4 B, PAO, 16.03 ± 2.69 clusters/100-µm axonal length; and PAO + B4-ECD, 29.77 ± 3.41 clusters/100-µm axonal length; Fig. 4 C, PAO, 0.29 ± 0.03 µm²; and PAO + B4-ECD, 0.34 ± 0.04 µm²). Based on our findings, we conclude that the increase in surface 7* nAChRs results from an increase in membrane insertion rather than a decrease in endocytosis.

View larger version (37K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Type III Nrg1 back signaling increases 7* nAChRs cluster area in the absence of endocytosis. Dissociated sensory neurons from E11 chick embryos were treated with B2-ECD (control) or B4-ECD for 1 h. In parallel, neurons were treated with PAO for 45 min and treated with B2-ECD or B4-ECD for an additional hour. Neurons were labeled with BgTx-488 (green), fixed, permeabilized, and colabeled for NF protein (blue). (A) Representative micrographs of BgTx-488 staining along NF-positive axons. B4-ECD treatment increased surface BgTx-488 clusters in the presence of PAO (d). Linescans of fluorescence intensity profiles of BgTx-488 along representative axons (see Materials and methods) are shown. Bar, 5 µm. (B) Quantification of surface BgTx-488 clusters along NF-labeled axons. B4-ECD treatment in the presence and absence of PAO induced 1.7 and 1.9 increases in surface BgTx-488 clusters along axons, respectively. Data were pooled from two independent experiments. The graph shows means ± SEM. Statistical significance was determined by ANOVA with post-hoc Fisher's PLSD test. *, P < 0.03 (Statview). (C) Quantification of surface BgTx-488 cluster area. B4-ECD treatment in the presence or absence of PAO induced an increase in BgTx-488 cluster area. Data pooled from two independent experiments were analyzed using nonparametric statistics and presented as box plots (see Materials and methods). Statistical significance was determined by the Kolmogorov-Smirnov Test. *, P = 0.03; **, P = 0.0001; ***, P < 0.0001 (Statview).

View larger version (42K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Type III Nrg1 back signaling activates the PtdIns 3K signaling pathway. (A) Dissociated sensory neurons from E11 chick embryos were treated for 5 min with B2-ECD (control), B4-ECD, 50 ng/ml NGF, or 10 ng/ml of soluble Nrg1β peptide (Nrg1-ECD). In parallel, neurons were treated with WM for 45 min before B4-ECD stimulation (WM + B4-ECD). Neurons were fixed, permeabilized, and costained for PIP3 (red) and tau protein (blue) to label axons. Both B4-ECD (g) and NGF (i) treatment induced puncta of PIP3 along tau-positive axons. Neither B4-ECD stimulation in the presence of WM (c and h) nor that of Nrg1-ECD (e and j) induced an increase in PIP3. Confocal images were obtained with a 40x oil objective. Bar, 10 µm. (B) Immunoblot analysis of phospho-Akt (Ser 473) in WT or Type III Nrg1–/– sensory neurons treated with either B2-ECD (control) or B4-ECD for 10 min. In WT neurons, B4-ECD treatment induced an approximately threefold increase in phospho-Akt, whereas no response was detected in mutant neurons. Total Akt in the bottom panel shows equal lysate loading. The bar graph represents phospho-Akt normalized to total Akt immunoreactive bands. Data are representative of three independent experiments. The graph shows means ± SEM. Statistical significance was determined by ANOVA with post-hoc Fisher's PLSD test. *, P < 0.002 (Statview). (C and D) E14.5 WT (a and b) or Type III Nrg1–/– (c and d) DRG explants were treated with B2-ECD (control) or B4-ECD for 10 min. Surface-bound B4-ECD or B2-ECD were labeled with an antibody against the human Fc domain (anti-Fc; green) before fixation. Neurons were fixed, permeabilized, and stained for phospho-Akt (red) and NF protein (blue). B4-ECD treatment increased phospho-Akt along Fc-positive axons of WT neurons (b and D) but did not do so along axons of mutant neurons (d). Note the close proximity of anti-FC and phospho-Akt puncta in the high-power micrograph shown in e. The asterisk denotes an axon negative for both anti-Fc and phospho-Akt immunolabeling (c). A 63x oil objective was used (a–d). Confocal imaging was obtained with a 100x oil objective (D). Bar: (a–d)10 µm; (D) 5 µm. (E) Quantification of the average fluorescence intensity (AFI) of phospho-Akt along axons of WT or Type III Nrg1–/– sensory neurons treated with B2-ECD (control) or B4-ECD for 10 min or 1, 2, or 6 h (see Materials and methods). Along WT axons, B4-ECD treatment induced increases in phospho-Akt. Along axons of mutant neurons, we did not detect an increase in phospho-Akt in response to B4-ECD treatment. The graph shows means ± SEM. Data are from three independent experiments. Statistical significance was determined by ANOVA. *, P < 0.02.

PtdIns 3K signaling activated by Type III Nrg1 back signaling is required for increased 7 nAChR surface expression
We tested whether PtdIns 3K activation is required for Type III Nrg1–mediated changes in 7* nAChR surface expression (Fig. 6). We treated dispersed sensory neurons with either B2-ECD (control) or B4-ECD for 1 h in the presence of 500 nM of the PtdIns 3K inhibitor WM. Surface 7* nAChR clusters were labeled with BgTx-488 (green), after which neurons were fixed and immunolabeled with an antibody against NF protein (blue; Fig. 6 A). In response to the B4-ECD treatment, we detected a significant increase in the number (Fig. 6 B) and area (Fig. 6 C) of BgTx-488 clusters along NF-positive processes. WM treatment alone did not affect the number of BgTx-488 clusters but did inhibit the response to B4-ECD, preventing the increase in the number of axonal surface 7*nAChRs (Fig. 6 B, B2-ECD [control], 9.08 ± 1.53 clusters/100-µm axonal length; B4-ECD, 17.41 ± 1.48 clusters/100-µm axonal length; WM, 9.58 ± 2.17 clusters/100-µm axonal length; and WM + B4-ECD, 10.75 ± 1.27 clusters/100-µm axonal length). Pretreatment with 5 µM of a small molecule inhibitor of Akt kinase activity (Akt inhibitor [Akt inh.]) also prevented the response to B4-ECD (Fig. 6 B, Akt inh., 10.33 ± 1.42 clusters/100-µm axonal length; and Akt inh. + B4-ECD, 8.48 ± 1.58 clusters/100-µm axonal length). Both WM and Akt inh. prevented the increase in surface BgTx cluster area in response to B4-ECD treatment (Fig. 6 C, B2-ECD [control], 0.25 ± 0.02 µm²; B4-ECD, 0.35 ± 0.02 µm²; WM, 0.22 ± 0.02 µm²; WM + B4-ECD, 0.23 ± 0.02 µm²; Akt inh., 0.22 ± 0.02 µm²; and Akt inh. + B4-ECD, 0.25 ± 0.02 µm²). We conclude that Type III Nrg1 back signaling activates PtdIns 3K signaling and that PtdIns 3K/Akt activities are required for Type III Nrg1–induced targeting of 7* nAChRs to the axonal surface.

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Figure 6. PtdIns 3K—Akt signaling activated by Type III Nrg1 back signaling is required for increased 7* nAChR surface expression. Dissociated sensory neurons from E11 chick embryos were treated with B2-ECD (control) or B4-ECD for 1 h. In parallel, neurons were pretreated with WM or an Akt inh. for 45 min before treatment with B2-ECD or B4-ECD for an additional hour. Neurons were labeled for surface 7* nAChRs with BgTx-488 (green), fixed, permeabilized, and costained for NF protein (blue). (A) Representative micrographs of BgTx-488 staining along NF-positive axons. B4-ECD treatment increased surface BgTx-488 clusters (b), which did not occur in the presence of WM (d). Linescans of fluorescence intensity profiles of BgTx-488 along representative axons (see Materials and methods) are shown. Bar, 5 µm. (B) Quantification of surface BgTx-488 clusters along sensory neuron axons represented in A. B4-ECD treatment induced an 1.9-fold increase of surface BgTx-488 clusters but not in the presence of WM or Akt inh. Data were pooled from three independent experiments. The graph shows means ± SEM. Statistical significance was determined by ANOVA with post-hoc Fisher's PLSD test. *, P < 0.0001 (Statview). (C) Quantification of surface BgTx-488 cluster area. B4-ECD treatment induced an increase in BgTx-488 cluster area but not in the presence of WM or Akt inh. Data pooled from three independent experiments were analyzed using nonparametric statistics and presented as box plots (see Materials and methods). Statistical significance determined by the Kolmogorov-Smirnov Test. *, P = 0.0001 (Statview).

	Discussion

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Prior work has shown that presynaptic Type III Nrg1 is critical for establishing stable interactions with postsynaptic partners and, ultimately, for the survival of neuromuscular synapses as well as for glia development and myelination (Wolpowitz et al., 2000; Michailov et al., 2004; Taveggia et al., 2005; Chen et al., 2006). Our studies demonstrate that Type III Nrg1 acts as a presynaptic signaling receptor. Activation of presynaptic Type III Nrg1 signaling, either by postsynaptic ErbB4 (as shown in Fig. S4, available at http://www.jcb.org/cgi/content/full/jcb.200710037/DC1), by the ErbB3 present on glia, or after depolarization (Bao et al., 2003, 2004), locally regulates the levels of presynaptic 7* nAChR surface expression. As a result, Type III Nrg1 back signaling, by regulating presynaptic 7* nAChRs, alters the magnitude of acetylcholine modulation of neurotransmitter release (MacDermott et al., 1999; Jones and Wonnacott, 2004).

We demonstrate that stimulating Type III Nrg1 activates a PtdIns 3K signaling pathway and that PtdIns 3K and Akt activity are required for increased surface expression of 7* nAChRs. At present, we do not know the molecular details of how Type III Nrg1 communicates with the PtdIns 3K signaling pathway or what Akt substrates regulate 7* nAChR trafficking. The intracellular domains of Type III Nrg1 lack known functional motifs that could mediate protein–protein interactions.

PtdIns 3K and its product PIP₃ are ubiquitous signaling molecules that link cell surface receptors to intracellular downstream effectors. PtdIns 3K signaling regulates the trafficking of several cell surface proteins, such as glucose transporter 4 (Tengholm and Meyer, 2002), excitatory amino acid carrier 1 glutamate transporter (Davis et al., 1998), calcium channels (Viard et al., 2004), and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (Man et al., 2003). An increased understanding of the molecular mechanisms that regulate trafficking of these proteins is certain to inform our further studies on presynaptic 7* nAChR targeting.

We have used BgTx binding to identify both surface and internal pools of 7* nAChRs. BgTx binding requires at least partial assembly of 7 subunits into pentamers (Mitra et al., 2001). Because we do not see effects of Type III Nrg1 back signaling on BgTx binding in permeabilized cells, it is unlikely that the early stages of assembling 7 subunits into functional receptors are targeted by back signaling. Assembly of 7* nAChRs and trafficking of 7* nAChRs through the exocytic pathway can be influenced by synaptic scaffolding proteins such as PICK1 (Baer et al., 2007), reversible palmitoylation (of 7* nAChRs by itself; Drisdel et al., 2004), tyrosine residues on unidentified proteins (Cho et al., 2005), and chronic nicotine (Marks et al., 1992). Any of these mechanisms might represent convergent targets with Type III Nrg1–PtdIns 3K signaling for influencing the rate of ER/Golgi to plasma membrane trafficking of 7* nAChRs.

Targeting nAChRs to presynaptic locales is an important part of establishing a plastic synapse. The 7* nAChRs in particular play a critical role in sustained modulation of neurotransmitter release (McGehee et al., 1995; Girod et al., 2000). Deficits in Type III Nrg1 signaling compromise the ability of nicotine to elicit sustained glutamatergic transmission at ventral hippocampal to nucleus accumbens synapses (Du, C., C. Zhong, M. Hancock, D.A. Talmage, and L.W. Role. 2004. Society for Neuroscience 34th Annual Meeting; unpublished data) and, based on the results presented here, we would predict a similar effect on nicotine/acetylcholine modulation of glutamate release from primary sensory afferents in the dorsal spinal cord (Genzen and McGehee, 2003). From the present data, we cannot say whether the requirement for Type III Nrg1 signaling is limited to developmental synaptogenesis or whether it continues to affect presynaptic 7* nAChR targeting to established synapses, contributing to the maintenance of synaptic plasticity throughout the lifespan. If Type III Nrg1 signaling does continue to regulate presynaptic levels of 7* nAChR or other receptors, then modulation of Type III Nrg1 signaling in sensory axons could contribute to adaptive physiological changes or to pathological changes in sensory transduction (Bermingham-McDonogh et al., 1997; Kerber et al., 2003).

There are interesting parallels between our findings and pathophysiological studies. Increasing evidence from genetic linkage studies has identified both the NRG1 (Stefansson et al., 2002; Harrison and Law, 2006) and the 7 nAChR gene CHRNA7 (Freedman et al., 2001; Leonard and Freedman, 2006) as susceptibility genes for schizophrenia. Postmortem studies have demonstrated decreased levels of BgTx binding in the hippocampus of schizophrenic patients (Freedman et al., 1995; Breese et al., 2000), and decreased levels of 7 mRNA and BgTx binding in dorsal lateral prefrontal cortex of schizophrenic brain are associated with risk alleles at the NRG1 locus (Mathew et al., 2007). It is worth noting that these risk alleles at the NRG1 locus also have been associated with increased expression of Type I and IV Nrg1 (Law et al., 2006). This raises the possibility that excess levels of soluble Nrg1 peptide disturbs the balance of bidirectional Nrg1-ErbB signaling that serves to ensure normal levels of functional 7* nAChRs.

	Materials and methods

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Animals and cell culture
DRG explants from E14.5 WT or Type III Nrg1^–/– mouse embryos (Wolpowitz et al., 2000) were dissected and cleaned with forceps to remove connective tissue. DRG were plated on glass coverslips (precoated with 1 mg/ml poly-D-lysine and 100 µg/ml laminin) and cultured in MEM (Invitrogen) supplemented with 10% FBS, 4 mg/ml glucose, 2 mM L-glutamine, 50 U/ml each of penicillin and streptomycin (Invitrogen), and 40 ng/ml β-NGF (Harlan Bioproducts for Science) at 37°C for 2 d. DRG from E11 chick embryos were dispersed and cultured in DME media (Invitrogen) supplemented with 5% chick embryonic extract, 10% horse serum, 2 mM L-glutamine, 50 U/ml penicillin and streptomycin (Invitrogen), and 50 ng/ml β-NGF (Harlan Bioproducts for Science) at 37°C for 2 d. For PIP₃ and phospho-Akt labeling and immunoblotting, culture media was replaced with serum-free media 4 h before soluble factor addition. For chick cultures, serum-free media consisted of DME, 1% BSA (Sigma-Aldrich), 2 mM L-glutamine (Invitrogen), insulin-transferrin-selenium supplement (Sigma-Aldrich) and 50 U/ml each of penicillin and streptomycin. For mouse cultures, serum-free media consisted of MEM, 1% BSA, 4 mg/ml glucose, 2 mM L-glutamine, insulin-transferrin-selenium supplement, and 50 U/ml each of penicillin and streptomycin.

Reagents
Where indicated, the following reagents were applied to the cells: 2 nM of soluble B2-ECD or B4-ECD (Bao et al., 2003), 50 ng/ml of recombinant HRG1-β1 EGF-like domain (R&D Systems), 50 ng/ml β-NGF (R&D Systems), 10 µg/ml CHX (Sigma-Aldrich), 5 µM phenylarsine oxide (Sigma-Aldrich), 500 nM WM (EMD), 5 µM Akt Inh. V (EMD), 2 µM each of PD 158780 and PD 168393 (EMD), and 200 nM TrkA inhibitor (EMD). Inhibitors were added to the media for 45 min before soluble factor addition.

Immunofluorescence
Cells were fixed with 4% PFA for 20 min at RT, permeabilized with 0.2% Triton X-100 (where indicated) for 5 min at RT, blocked with 10% NDS, and incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: anti-Type III Nrg1 (1:500; Yang et al., 1998), anti-NF (1:500; Sternberger Monoclonals, Inc.), anti-tau (1:250; Sigma-Aldrich), anti-human IgG (Fc-specific; 1:200; Sigma-Aldrich), anti-PI_3,4,5P₃ (1:50; Echelon Biosciences Inc.), anti–phospho-Akt (1:200; Cell Signaling Technology), anti-synaptophysin (1:500; Millipore); anti-vGlut1 (1:200; Synaptic Systems GmbH), anti-TrkA (1:5,000; gift from L.F. Reichardt, University of California, San Francisco, San Francisco, CA), anti-MAP2 (1:500; Millipore), and anti–phospho MAPK (1:1,000; Sigma-Aldrich). Cells were washed and incubated in secondary antibodies conjugated to Alexa 488 (1:500; Invitrogen), Alexa 594 (1:500; Invitrogen), or anti-mannobioside carbohydrate antibodies (1:50; Jackson ImmunoResearch Laboratories) for 1 h at RT. To label PI_3,4,5P₃, slips were treated with Avidin/Biotin Blocking kit (Vector Laboratories) before incubation in anti-PI_3,4,5P₃. The primary antibody signal was amplified by sequential binding with a biotinylated anti–mouse secondary (1:200; Jackson ImmunoResearch Laboratories) and NeutrAvidin–-rhodamine red (1:200; Invitrogen). Slips were mounted using VectaShield (Vector Laboratories), and images were captured using a microscope (Axio Imager; Carl Zeiss, Inc.) equipped with Plan-Apochromat objectives (20x with 0.8 NA or 63x oil with 1.4 NA), a charge-coupled device camera (Hamamatsu), and Metamorph software (Version 6.3r5; MDS Analytical Technologies). Confocal images were captured with an NLO Multiphoton (LSM 510; Carl Zeiss, Inc.) on a microscope (Axioskop2 FS; Carl Zeiss, Inc.) equipped with Plan-Neofluar objectives (40 or 100x oil with 1.3 NA) and a charge-coupled device camera. Brightness and contrast were adjusted using Photoshop software (Version 8.0; Adobe).

To label surface 7* nAChRs, live sensory neurons were incubated in BgTx-488 (1:500; Invitrogen) for 15–20 min at 37°C. Cells were fixed and incubated in rabbit anti–Alexa 488 (1:250; Invitrogen) overnight at 4°C, followed by secondary antibody incubation. Surface cluster number and area were measured along NF-positive processes (10 µm from soma) using Metamorph software. The lengths of axonal processes were measured by manually tracing NF-positive processes. Control cultures in each experiment were used to define the threshold for measuring clusters at 50% maximum intensity and greater than or equal to four contiguous pixels (Kawai et al., 2002). To determine nonspecific binding, cultures were treated with 1 µM of nicotine or 5 µM methyllycaconitine before labeling. For each experiment, nonspecific labeling was 12% and was subtracted from all counts. Linescans with widths of 10 contiguous pixels were obtained using Metamorph software.

To quantify Akt activation along axons, neurons were stained with antibodies against phospho-Akt and NF to label axons. 15–20 axons per condition were analyzed in each experiment. The AFI of phospho-Akt along NF-positive processes was measured using MetaMorph software. The AFI of two to three randomly selected regions for each condition were measured as background AFI and subtracted from all measurements. To compare results between independent experiments, the mean AFI from different conditions in each independent experiment was normalized to the control AFI and is expressed as fold increase.

Ca²⁺ imaging
After 3 d in vitro, DRG explants were rinsed with HBSS, loaded with Fluo-3 Ca²⁺ binding dye (Invitrogen) dissolved in HBSS for 30 min at 37°C, and rinsed for 30 min at 37°C in HBSS. The coverslip was placed on a perfusion-equipped stage and perfused at a rate of 0.5 ml/min with HBSS containing 2 µM tetrodotoxin, 10 µM bicuculin, 50 µM D-(–)-2amino-5-phosphono-valeric acid and 20 µM CNQX (Sigma-Aldrich). Images were captured every 5 s using a spinning disc confocal microscope (DSU; Olympus) equipped with a UPlanSApo objective (60x water with 1.4 NA), electron-multiplying charge-coupled device camera (Hamamatsu), and Slidebook software (Version 4; Olympus). After acquiring images for 1 min, 1 µM of nicotine was focally applied by pressure ejection for 60 s and the explants were imaged for another 4 min. After a 1-µM nicotine application, fluorescence intensity within defined axonal regions was quantified with MetaMorph software.

¹²⁵I-BgTx binding assay
After 2 d in vitro, dispersed chick sensory neurons were treated with 2 nM of soluble B2-ECD or B4-ECD for 24 h. 6 nM ¹²⁵I-BgTx (2,000 Ci/µmol; GE Healthcare) was added directly to the media for the final 20 min. To measure total ¹²⁵I-BgTx binding, cultures were rinsed three times with PBS and permeabilized with 0.5% saponin in 0.2% FAF-BSA/PBS for 30 min before incubation with 6 nM ¹²⁵I-BgTx in 0.2% FAF-BSA/PBS for 2 h on ice. For both surface and total labeling, nonspecific binding was assessed by including 1 µM BgTx (Sigma-Aldrich) during the labeling with ¹²⁵I-BgTx. After labeling, cells were washed and solubilized in 1 N NaOH, and bound ¹²⁵I-BgTx was quantified with a counter. Nonspecific binding was subtracted.

Immunoblotting
Neurons were lysed in RIPA buffer (Thermo Fisher Scientific) supplemented with 10 mM DTT, protease inhibitors (Sigma-Aldrich), and phosphatase inhibitors (Thermo Fisher Scientific). Lysates (phospho-Akt, 25 µg; 7, 40 µg) were separated on 10% SDS-PAGE gels and transferred to nitrocellulose filters. Filters were blocked in 5% milk in 0.1% Tween-20 TBS solution at RT for 2 h before overnight incubation in primary antibody solutions in 5% BSA or milk in Tween-20 TBS solution at 4°C (anti-7 nAChR subunit [1:500; Santa Cruz Biotechnology, Inc.], anti-MAPK [1:5,000; Millipore], anti-glyceraldehyde 3-phosphate dehydrogenase [1:10,000; Millipore], anti–NF M [1:7,500; Millipore), anti–phospho-Akt [1:500; Cell Signaling Technology], and anti-Akt [1:1,000; Cell Signaling Technology]). Detection was performed using Alexa Fluor 680 (Invitrogen) or IRDye-800 (Rockland Immunochemicals) secondary antibodies and an Odyssey Infrared Imaging System (Version 2.1; LI-COR Biosciences).

Transferrin recycling
After 2–4 d in vitro, WT or Type III Nrg1^–/– sensory neurons were serum starved for 25 min in serum-free MEM containing 0.5% BSA. Neurons were incubated with 10 µg/ml transferrin–Alexa 488 (Invitrogen) for 15 min in serum-free media, after which cells were rinsed and chased in complete media supplemented with 1 mg/ml of unlabeled transferrin (Sigma-Aldrich). During the chase period, live images of neurons were captured at 0.5–1-µm intervals every 2–4 min over a >20-min period using a spinning disc confocal microscope (DSU; Olympus) equipped with a UPlanSApo objective (60x water with 1.4 NA), electron-multiplying charge-coupled device camera, and Slidebook software. The AFI of transferrin–Alexa 488 in collapsed z series was measured using MetaMorph software. The AFI of two to three randomly selected regions for each condition was measured as background AFI and subtracted. The AFI from each time point was normalized to the AFI at t = 0 and is expressed as percentage of initial AFI.

Statistical significance
For normally distributed data, statistical significance was evaluated by ANOVA with a post-hoc Fischer's PLSD test for multiple comparisons (Statview; Adept Scientific). Non-normally distributed data were analyzed using nonparametric methods and are presented using box plots. The boxes include data points within the middle 50%. The bottom marks the twenty-fifth percentile, the middle line the fiftieth percentile, and the top the seventy-fifth percentile. Vertical lines mark the fifth and ninety-fifth percentiles (see Devay et al. [1999] for a detailed description of data presentation). Statistical significance was evaluated by the Kolmogorov-Smirnov test.

Online supplemental material
In Fig. S1, we show that endocytic recycling of transferrin (A and B) and the trafficking of presynaptic proteins vGlut1 and synaptophysin (C and D) are not impaired in Type III Nrg1^–/– sensory neurons. Additionally, we show that activation of Type III Nrg1 back-signaling does not induce an increase in the surface expression of TrkA along axons (E and F). Fig. S2 shows soluble ErbB4-ECD (B4-ECD) bound to Type III Nrg1 puncta along WT axons only (A). B4-ECD treatment induces an increase in the axonal surface expression of 7*nAChRs in a dose-dependent manner (B). Fig. S3 shows that Type III Nrg1 back signaling activates Akt in neurons and not nonneuronal cells (A). Inhibition of ErbB (B) or TrkA (C) kinase activity does not prevent the B4-ECD-induced response. We also show that B4-ECD treatment does not activate MAPK in neurons (D). Fig. S4 consists a diagram summarizing a mechanism by which Type III Nrg1 back-signaling regulates the levels of 7*nAChRs along axons. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.200710037/DC1.

	Acknowledgments

 
We thank M. Mertz for technical assistance with Ca²⁺ imaging, B.L. Sherman for mouse genotyping, M.A. Johnson for helpful comments on the manuscript, and L.F. Reichardt for the TrkA antibody.

This work was funded by grants from the National Institutes of Health (NS29071 to L.W. Role and D.A. Talmage; DA019941 to L.W. Role; and DK07328 to M.L. Hancock) and from the National Alliance for Research on Schizophrenia and Depression (Grable Distinguished Investigator Award to L.W. Role).

Submitted: 4 October 2007

Accepted: 31 March 2008

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