The neuregulin-1 receptor erbB4 controls glutamatergic synapse maturation and plasticity

Neuregulin-1 (NRG1) signaling participates in numerous neurodevelopmental processes. Through linkage analysis, nrg1 has been associated with schizophrenia, although its pathophysiological role is not understood. The prevailing models of schizophrenia invoke hypofunction of the glutamatergic synapse and defects in early development of hippocampal-cortical circuitry. Here, we show that the erbB4 receptor, as a postsynaptic target of NRG1, plays a key role in activity-dependent maturation and plasticity of excitatory synaptic structure and function. Synaptic activity leads to the activation and recruitment of erbB4 into the synapse. Overexpressed erbB4 selectively enhances AMPA synaptic currents and increases dendritic spine size. Preventing NRG1/erbB4 signaling destabilizes synaptic AMPA receptors and leads to loss of synaptic NMDA currents and spines. Our results indicate that normal activity-driven glutamatergic synapse development is impaired by genetic deficits in NRG1/erbB4 signaling leading to glutamatergic hypofunction. These findings link proposed effectors in schizophrenia: NRG1/erbB4 signaling perturbation, neurodevelopmental deficit, and glutamatergic hypofunction.     

Patterning of muscle acetylcholine receptor gene expression in the absence of motor innervation

The patterning of skeletal muscle is thought to depend upon signals provided by motor neurons. We show that AChR gene expression and AChR clusters are concentrated in the central region of embryonic skeletal muscle in the absence of innervation. Neurally derived Agrin is dispensable for this early phase of AChR expression, but MuSK, a receptor tyrosine kinase activated by Agrin, is required to establish this AChR prepattern. The zone of AChR expression in muscle lacking motor axons is wider than normal, indicating that neural signals refine this muscle-autonomous prepattern. Neuronal Neuregulin-1, however, is not involved in this refinement process, nor indeed in synapse-specific AChR gene expression. Our results demonstrate that AChR expression is patterned in the absence of innervation, raising the possibility that similarly prepatterned muscle-derived cues restrict axon growth and initiate synapse formation.     

Synapse-specific expression of acetylcholine receptor genes and their products at original synaptic sites in rat soleus muscle fibres regenerating in the absence of innervation.

To test the hypothesis that synaptic basal lamina can induce synapse-specific expression of acetylcholine receptor (AChR) genes, we examined the levels mRNA for the alpha- and epsilon-subunits of the AChR in regenerating rat soleus muscles up to 17 days of regeneration. Following destruction of all muscle fibres and their nuclei by exposure to venom of the Australian tiger snake, new fibres regenerated within the original basal lamina sheaths. Northern blots showed that original mRNA was lost during degeneration. Early in regeneration, both alpha- and epsilon-subunit mRNAs were present throughout the muscle fibres but in situ hybridization showed them to be concentrated primarily at original synaptic sites, even when the nerve was absent during regeneration. A similar concentration was seen in denervated regenerating muscles kept active by electrical stimulation and in muscles frozen 41-44 hours after venom injection to destroy all cells in the synaptic region of the muscle. Acetylcholine-gated ion channels with properties similar to those at normal neuromuscular junctions were concentrated at original synaptic sites on denervated stimulated muscles. Taken together, these findings provide strong evidence that factors that induce the synapse-specific expression of AChR genes are stably bound to synaptic basal lamina.     

The signaling pathways mediated by P2Y nucleotide receptors in the formation and maintenance of the skeletal neuromuscular junction

The motor neuron, the Schwann cell and the muscle cell are highly specialized at the vertebrate skeletal neuromuscular junction (NMJ). The muscle cell surface contains a high local density of acetylcholine (ACh) receptors (AChRs), acetylcholinesterase (AChE) and their interacting macromolecules at the NMJ, forming the postsynaptic specializations. During the early stages of development, the incoming nerve terminal induces the formation of these postsynaptic specializations; the nerve secretes agrin and neuregulin (NRG), which are known to aggregate existing AChRs and to increase the expression of AChR at the synaptic region, respectively. In addition, adenosine 5'-triphosphate (ATP) is stored at the motor nerve terminals and is coreleased with ACh during muscle contraction. Recent evidence suggests that ATP can play a role in forming and maintaining the postsynaptic specializations by activating its corresponding receptors. In particular, one of the nucleotide receptor subtypes, the P2Y(1) receptor, is specifically localized at the NMJs. The gene expression of AChR and AChE is upregulated after the activation of P2Y(1) receptors. Thus, the synaptic ATP together with agrin and NRG can act as a synapse-organizing factor to induce the expression of postsynaptic functional effectors.     

Developmental Regulation of Neuregulin1 Isoforms and erbB Receptor Expression in Intact Rat Dorsal Root Ganglia

Neuregulins (NRGs) are a family of growth factors which bind to the erbB family of tyrosine kinase receptors. The exact nature and interaction of specific NRG isoforms and erbB receptors that occur during the development of the nervous system have not been reported. In order to better understand the role that different NRG isoforms and erbB receptors play in the differentiation, proliferation, and survival of neurons and glial cells, we isolated protein and mRNA from dorsal root ganglia of rat pups between embryonic day (E) 13 and postnatal day (P) 15. The relative expression levels of the NRGs and erbB receptors for the different time points were compared using both Western and RT-PCR analyses. NRG1-type1α protein levels were highest at E-13 and then decreased by approximately 40% and remained constant through P-15. In contrast, mRNA levels for NRG1-type1α remained constant from E-15 to P-15. The protein levels for NRG1-type 1β were similar to NRG1-type1α at E-13 with an approximate 40% increase in the levels at E-15 and E-17 followed by a decrease to E-13 levels for the remainder of the developmental time periods. The mRNA levels for NRG1-type1β remained constant from E-15 to P-15. The protein and mRNA expression patterns for each erbB receptor were distinctive. The protein levels for erbB-2 were highest at E-19 while erbB-3 levels were highest at E-17 and E-18. ErbB-4 protein levels were highest at E-13 and decreased through P-15. The developmental pattern for erbB-2 and erbB-4 mRNA levels had no relation to that of the corresponding protein levels while the mRNA levels for erbB-3 were highest at E-17 and E-18 similar to the pattern observed for the erbB-3 protein levels. We concluded that both NRG and erbB expression in dorsal root ganglia are mostly translationally controlled and that NRG1 isoforms and their erbB receptors are not coordinately regulated. Special issue article in honor of Dr. George DeVries.     

A novel pathway for MuSK to induce key genes in neuromuscular synapse formation

At the developing neuromuscular junction the Agrin receptor MuSK is the central organizer of subsynaptic differentiation induced by Agrin from the nerve. The expression of musk itself is also regulated by the nerve, but the mechanisms involved are not known. Here, we analyzed the activation of a musk promoter reporter construct in muscle fibers in vivo and in cultured myotubes, using transfection of multiple combinations of expression vectors for potential signaling components. We show that neuronal Agrin by activating MuSK regulates the expression of musk via two pathways: the Agrin-induced assembly of muscle-derived neuregulin (NRG)-1/ErbB, the pathway thought to regulate acetylcholine receptor (AChR) expression at the synapse, and via a direct shunt involving Agrin-induced activation of Rac. Both pathways converge onto the same regulatory element in the musk promoter that is also thought to confer synapse-specific expression to AChR subunit genes. In this way, a positive feedback signaling loop is established that maintains musk expression at the synapse when impulse transmission becomes functional. The same pathways are used to regulate synaptic expression of AChR epsilon. We propose that the novel pathway stabilizes the synapse early in development, whereas the NRG/ErbB pathway supports maintenance of the mature synapse.     

Extracellular domains drive homo- but not hetero-dimerization of erbB receptors

Many different growth factor ligands, including epidermal growth factor (EGF) and the neuregulins (NRGs), regulate members of the erbB/HER family of receptor tyrosine kinases. These growth factors induce erbB receptor oligomerization, and their biological specificity is thought to be defined by the combination of homo- and hetero-oligomers that they stabilize upon binding. One model proposed for ligand-induced erbB receptor hetero-oligomerization involves simple heterodimerization; another suggests that higher order hetero-oligomers are 'nucleated' by ligand-induced homodimers. To distinguish between these possibilities, we compared the abilities of EGF and NRG1-beta1 to induce homo- and hetero-oligomerization of purified erbB receptor extracellular domains. EGF and NRG1-beta1 induced efficient homo-oligomerization of the erbB1 and erbB4 extracellular domains, respectively. In contrast, ligand-induced erbB receptor extracellular domain hetero-oligomers did not form (except for s-erbB2-s-erbB4 hetero-oligomers). Our findings argue that erbB receptor extracellular domains do not recapitulate most heteromeric interactions of the erbB receptors, yet reproduce their ligand-induced homo-oligomerization properties very well. This suggests that mechanisms for homo- and hetero-oligomerization of erbB receptors are different, and contradicts the simple heterodimerization hypothesis prevailing in the literature.     

Neuregulin-heparan-sulfate proteoglycan interactions produce sustained erbB receptor activation required for the induction of acetylcholine receptors in muscle

Neuregulins bind to and activate members of the EGF receptor family of tyrosine kinases that initiate a signaling cascade that induces acetylcholine receptor synthesis in the postsynaptic membrane of neuromuscular synapses. In addition to an EGF-like domain, sufficient for receptor binding and tyrosine auto-phosphorylation, many spliced forms also have an IG-like domain that binds HSPGs and maintains a high concentration of neuregulin at synapses. Here, we show that the IG-like domain functions to keep the EGF-like domain at sufficiently high concentrations for a sufficiently long period of time necessary to induce acetylcholine receptor gene expression in primary chick myotubes. Using recombinant neuregulins with and without the IG-like domain, we found that IG-like domain binding to endogenous HSPGs produces a 4-fold increase in receptor phosphorylation. This enhancement of activity was blocked by soluble heparin or by pretreatment of muscle cells with heparitinase. We show that at least 12-24 h of neuregulin exposure was required to turn on substantial acetylcholine receptor gene expression and that the erbB receptors need to be kept phosphorylated during this time. The need for sustained erbB receptor activation may be the reason why neuregulins are so highly concentrated in the extracellular matrix of synapses.     

NRG1 and synaptic function in the CNS

Recent genetic evidence indicates that neuregulin 1 (NRG1) and its receptor erbB4 may be susceptibility genes in schizophrenia, but their function in CNS synaptic transmission and circuitry is not well understood. In this issue of Neuron, studies from Li et al. and Woo et al. show that NRG1 and erbB4 regulate transmission at brain glutamate and GABA synapses. These findings raise the possibility of synaptic defects in schizophrenia.     

Intermittent hypoxia induces the proliferation of rat vascular smooth muscle cell with the increases in epidermal growth factor family and erbB2 receptor

Obstructive sleep apnea is characterized by intermittent hypoxia (IH), and associated with cardiovascular diseases, such as stroke and heart failure. These cardiovascular diseases have a relation to atherosclerosis marked by the proliferation of vascular smooth muscle cells (VSMCs). In this study, we investigated the influence of IH on cultured rat aortic smooth muscle cell (RASMC). The proliferation of RASMC was significantly increased by IH without changing the level of apoptosis. In order to see what induces RASMC proliferation, we investigated the influence of normoxia (N)-, IH- and sustained hypoxia (SH)-treated cell conditioned media on RASMC proliferation. IH-treated cell conditioned medium significantly increased RASMC proliferation compared with N-treated cell conditioned medium, but SH-treated cell conditioned medium did not. We next investigated the epidermal growth factor (EGF) family as autocrine growth factors. Among the EGF family, we found significant increases in mRNAs for epiregulin (ER), amphiregulin (AR) and neuregulin-1 (NRG1) in IH-treated cells and mature ER in IH-treated cell conditioned medium. We next investigated the changes in erbB family receptors that are receptors for ER, AR and NRG1, and found that erbB2 receptor mRNA and protein expressions were increased by IH, but not by SH. Phosphorylation of erbB2 receptor at Tyr-1248 that mediates intracellular signaling for several physiological effects including cell proliferation was increased by IH, but not by SH. In addition, inhibitor for erbB2 receptor suppressed IH-induced cell proliferation. These results provide the first demonstration that IH induces VSMC proliferation, and suggest that EGF family, such as ER, AR and NRG1, and erbB2 receptor could be involved in the IH-induced VSMC proliferation.