Acetylcholine Receptor-inducing Activity Research Articles

Proteolytic processing of Neuregulin-1.

Neuregulin-1 (NRG1), known also as heregulin, acetylcholine receptor inducing activity (ARIA), glial growth factor (GGF), or sensory and motor neuron derived factor (SMDF), is a key factor for many developmental processes and in adult brain. All known splice variants contain an epidermal growth factor (EGF)-like domain, which is mediating signaling via receptors of the ErbB family. In particular, NRG1 acts as an essential signaling molecule expressed on the axonal surface, where it signals to Schwann cells throughout development and regulates the thickness of the myelin sheath. NRG1 is required also by other cell types in the nervous system, for instance as an axonal signal released by proprioceptive afferents to induce development of the muscle spindle, and it controls aspects of cortical interneuron development as well as the formation of thalamo-cortical projections. The precursor protein of NRG1 can be activated and released from the membrane through limited proteolysis by the β-Secretase (β-site amyloid precursor protein cleaving enzyme 1, BACE1) which was first identified through its function as the rate limiting enzyme of amyloid-β-peptide (Aβ) production. Aβ is the major component of amyloid plaques in Alzheimer's disease (AD). Due to the hairpin nature of NRG1 type III two membrane-bound stubs with a type 1 and a type 2 orientation are generated by an initial proteolytic cleavage and successive release of the EGF-like domain either by dual cleavage by BACE1 or by ADAM17 (a disintegrin and metalloprotease) which is also called TACE (Tumor Necrosis Factor-α-converting enzyme). The cleavages activate NRG1 to allow juxtacrine or paracrine signaling. The type 1 oriented stub is further cleaved by γ-secretase in the transmembrane domain with a putative role in intracellular domain (ICD) signaling, while the type II oriented stub is cleaved by signal peptidase like proteases (SPPLs). Neuregulin-1 was identified as a major physiological substrate of BACE1 during early postnatal development when similarities in BACE1 KO mice and NRG1 heterozygous mice were discovered. Both display severe hypomyelination of peripheral nerves. Later it was shown with genetic and pharmacological evidence that the developmental effect of type I NRG1 on the formation and the maintenance of muscle spindles is BACE1 dependent. Thus, NRG1 functions in PNS and CNS are likely to set limits to an Alzheimer disease therapy with relatively strong BACE1 inhibition.

BACE1 Dependent Neuregulin Processing: Review

Neuregulin-1 (NRG1), known also as heregulin, acetylcholine receptor inducing activity (ARIA), glial growth factor (GGF), or sensory and motor neuron derived factor (SMDF), plays essential roles in several developmental processes, and is required also later in life. Many variants of NRG1 are produced via alternative splicing and usage of distinct promoters. All contain an epidermal growth factor (EGF)-like domain, which alone is sufficient to bind and activate the cognate receptors, members of the ErbB family. NRG1 mediated signaling is crucial for cardiogenesis and the development of the mammary gland and ErbB2 (HER2), an orphan co-receptor for NRG1 is the target of the drug Herceptin� (trastuzumab) used for treatment of metastatic breast cancer. In the nervous system, NRG1 controls the early development of subpopulations of neural crest cells. In particular, NRG1 acts as an essential paracrine signaling molecule expressed on the axonal surface, where it signals to Schwann cells throughout development and regulates the thickness of the myelin sheath. NRG1 is required also by other cell types in the nervous system, for instance as an axonal signal released by proprioceptive afferents to induce development of the muscle spindle, and it controls aspects of cortical interneuron development as well as the formation of thalamocortical projections. Work from several laboratories implicates dysregulation of NRG1/ErbB4 signaling in the etiology of schizophrenia. Biochemical studies have shown that the precursor proteins of NRG1 can be released from the membrane through limited proteolysis. In addition, most NRG1 isoforms contain a transmembrane domain, which is processed by γ-secretase after shedding. Thereby the intracellular domain is released into the cytoplasm. Despite this, the importance of NRG1 cleavage for its functions in vivo remained unclear until recently. β- Secretase (β-site amyloid precursor protein cleaving enzyme 1, BACE1) was first identified through its function as the rate limiting enzyme of amyloid-β-peptide (Aβ) production. Aβ is the major component of amyloid plaques in Alzheimer's disease (AD). More recently it was shown that Neuregulin-1 is a major physiological substrate of BACE1 during early postnatal development. Mutant mice lacking BACE1 display severe hypomyelination of peripheral nerves similar to that seen in mice lacking NRG1/ErbB signaling in Schwann cells, and a BACE1-dependent activation of NRG1 in the process of peripheral myelination was proposed. Here we summarize the current knowledge about the role of NRG1 proteolysis for ErbB receptor mediated signaling during development and in Alzheimer's disease.

CATALYTIC RECEPTORS

CATALYTIC RECEPTORS

Neuregulins regulate cardiac parasympathetic activity: muscarinic modulation of beta-adrenergic activity in myocytes from mice with neuregulin-1 gene deletion.

Neuregulins are required for maintenance of acetylcholine receptor-inducing activity of nicotinic receptors in neurons and skeletal muscle, but effects of neuregulins on muscarinic receptors are not known. In the normal heart, parasympathetic activation counterbalances beta-adrenergic activation. To test the hypothesis that neuregulins modify parasympathetic function in the heart, we studied cardiomyocytes from mice heterozygous for neuregulin-1 gene deletion (NRG-1+/-) and examined the effects of beta-adrenergic stimulation on contractility in the presence and absence of the muscarinic agonist carbachol. We evaluated contraction and intracellular Ca2+ transients ([Ca2+]i) in left ventricular (LV) myocytes loaded with Fluo-3 from NRG-1+/- and wild-type (WT) mice. Under baseline conditions (0.5 Hz, 1.5 mmol/L [Ca2+]o, 25 degrees C), characteristics of myocyte contraction/relengthening and systolic/diastolic [Ca2+]i were not different between WT and NRG-1+/- mice. The steady-state increases in fractional shortening (FS) and peak-systolic [Ca2+]i in response to isoproterenol were similar in both groups. In WT myocytes stimulated with isoproterenol, carbachol decreased FS, peak-systolic [Ca2+]i, and cAMP levels. In NRG-1+/- myocytes, carbachol did not attenuate either FS or peak-systolic [Ca2+]i, associated with the failure to decrease cAMP levels. Investigation of muscarinic receptor signaling showed no difference of LV protein levels of muscarinic M2 receptors or G protein Galpha(i1,2), Galpha(i3), and Galpha(o) subunits. Cardiomyocytes deficient in neuregulin signaling are unable to adequately counterbalance beta-adrenergic activation by inhibitory parasympathetic activity. This mechanism may contribute to the known increased risk of heart failure in injured human hearts when neuregulin signaling is suppressed.

Neuregulins and the neuromuscular system: 10 years of answers and questions.

The neuregulins were originally discovered in searches for the acetylcholine receptor-inducing activity (ARIA), glial growth factor (GGF), and a ligand for the oncogene neu (ErbB2/HER2). Neuregulin1 (NRG1)-mediated cell communication is critical in the central and peripheral nervous system, heart, breast, and other organ systems. This review will focus on the functions of NRG1s in the development and maintenance of the neuromuscular system and on the regulation of NRG1 signaling within this system. The roles of NRG1 signaling in the neuromuscular system are far more pervasive than contemplated when neuregulins were discovered 10 years ago. In fact, neuregulin-mediated cell communication plays an essential role in the biology of most components of the neuromuscular system--including motor and sensory neurons, muscle fibers, Schwann cells, and major specializations (neuromuscular synapses, muscle spindles, Golgi tendon organs, and peripheral nerves). It is argued here that while NRG1 proteins are indeed "ARIA" and "GGF", their involvement in regulating synapse-specific transcription and Schwann cell development is more complex than originally proposed. It is also argued that NRG1 isoforms differ in their signaling properties and that these differences tailor specific isoforms for specific signaling tasks; for example, some NRG1 isoforms may be specialized for paracrine signaling and others for juxtacrine signaling. In the first 10 years of neuregulin research there has been much progress in understanding the actions of neuregulins in shaping and maintaining the neuromuscular system. However, major questions, old and new, remain unanswered; and the second 10 years promises to be at least as exciting as the first.

Developmental Expression of Muscarinic Acetylcholine Receptors in Chick Retina: Selective Induction of M2Muscarinic Receptor ExpressionIn Ovoby a Factor Secreted by Müller Glial Cells

Muscarinic acetylcholine receptors (mAChRs) play an important role in signal processing in the retina. We have used subtype-specific antibodies to identify the changes in the localization of mAChR expression during embryonic development of the retina in vivo and their relationship to the changes in mAChRs in retinal cells in culture. We have demonstrated previously that treatment of fresh retinal cultures with conditioned media from mature retinal cultures specifically induces expression of the M(2) mAChR (McKinnon et al., 1998). We show that the M(2)-inducing activity, which we tentatively have called MARIA (muscarinic acetylcholine receptor-inducing activity) is produced by Müller glial cells in culture, because significant activity can be found in media conditioned by essentially neuron-free cultures of Müller glia, as well as by a Müller glial cell line but not several neuroblastoma cell lines. We also demonstrate that the appearance of the M(2) receptor in vivo occurs concomitantly with the appearance of significant numbers of Müller glial cells in the developing retina. Furthermore, the administration of crude or partially purified preparations of MARIA to developing chick embryos in ovo induces precocious expression of M(2) mAChRs in the appropriate cell types in the retina. These results show that a factor secreted by cultured retinal Müller glia can regulate M(2) mAChR expression in vivo and in vitro and suggest that the secretion of MARIA by Müller glia in vivo may be responsible for the normal induction of M(2) mAChR expression during embryonic development.

The Release of Acetylcholine Receptor Inducing Activity (ARIA) from Its Transmembrane Precursor in Transfected Fibroblasts

Acetylcholine receptor inducing activity (ARIA) is made by motoneurons and is released at the neuromuscular synapse to stimulate the synthesis of acetylcholine receptors by skeletal muscle. ARIA is derived from a transmembrane precursor (pro-ARIA) via proteolytic cleavage of the ectodomain. We studied requirements in the amino acid sequence at the cleavage site with various substitution and deletion mutations. Wild type (WT) and mutant proteins were transiently expressed in COS cells, and release of ARIA into the conditioned medium was measured by tyrosine phosphorylation of its receptor, p185, in L6 cells. Removal of all potential cleavage sites between the extracellular epidermal growth factor domain and the transmembrane domain by substitution and small deletions (<11 amino acid residues out of 21) did not significantly reduce ARIA release, whereas larger deletions abolished it. We propose that cleavage occurs independently of amino acid sequence at a short distance from the epidermal growth factor domain, unless sterically hindered by the nearby secondary structure. A mutant with shorter cytoplasmic domain ("c" isoform) released significantly less ARIA than the WT ("a" isoform), suggesting that the c isoform may be suitable for signaling through direct cell-cell contact. Alternatively, proteolytic conversion of the a isoform to the c isoform may rapidly down-regulate release of ARIA.

Processing of ARIA and release from isolated nerve terminals.

The neuromuscular junction is a specialized synapse in that every action potential in the presynaptic nerve terminal results in an action potential in the postsynaptic membrane, unlike most interneuronal synapses where a single presynaptic input makes only a small contribution to the population postsynaptic response. The postsynaptic membrane at the neuromuscular junction contains a high density of neurotransmitter (acetylcholine) receptors and a high density of voltage-gated Na+ channels. Thus, the large acetylcholine activated current occurs at the same site where the threshold for action potential generation is low. Acetylcholine receptor inducing activity (ARIA), a 42 kD protein, that stimulates synthesis of acetylcholine receptors and voltage-gated Na+ channels in cultured myotubes, probably plays the same roles at developing and mature motor endplates in vivo. ARIA is synthesized as part of a larger, transmembrane, precursor protein called proARIA. Delivery of ARIA from motor neuron cell bodies in the spinal cord to the target endplates involves several steps, including proteolytic cleavage of proARIA. ARIA is also expressed in the central nervous system and it is abundant in the molecular layer of the cerebellum. In this paper we describe our first experiments on the processing and release of ARIA from subcellular fractions containing synaptosomes from the chick cerebellum as a model system.

The Neuregulin Precursor proARIA Is Processed to ARIA after Expression on the Cell Surface by a Protein Kinase C-Enhanced Mechanism

We have investigated how the transmembrane precursor proARIA is processed to ARIA (acetylcholine receptor-inducing activity). Pulse-chase labeling in transfected Chinese hamster ovary (CHO) cells showed that proARIA was cleaved to release ARIA into the medium. Cell surface biotin-labeling experiments demonstrated that proARIA was first expressed on the cell surface before being rapidly cleaved to release biotin-labeled ARIA into the medium. While not essential for proteolytic cleavage of proARIA, serum or phorbol-12-myristate-13-acetate (PMA), which activates protein kinase C (PKC), was needed for the efficient release of the processed ARIA. Proteolytic cleavage was blocked by brefeldin A, suggesting that processing occurred distal to Golgi compartments, and by NH4Cl, suggesting a need for intracellular acidic compartments. Serum and PMA also stimulated ARIA release from cultured sensory neurons, suggesting that a similar regulated release mechanism occurs in neurons and may be important in determining where ARIA is released in the developing nervous system.

Neuregulins Promote Survival and Growth of Cardiac Myocytes: PERSISTENCE OF ErbB2 AND ErbB4 EXPRESSION IN NEONATAL AND ADULT VENTRICULAR MYOCYTES

Neuregulins (i.e. neuregulin-1 (NRG1), also called neu differentiation factor, heregulin, glial growth factor, and acetylcholine receptor-inducing activity) are known to induce growth and differentiation of epithelial, glial, neuronal, and skeletal muscle cells. Unexpectedly, mice with loss of function mutations of NRG1 or of either of two of their cognate receptors, ErbB2 and ErbB4, die during midembryogenesis due to the aborted development of myocardial trabeculae in ventricular muscle. To examine the role of NRG and their receptors in developing and postnatal myocardium, we studied the ability of a soluble NRG1 (recombinant human glial growth factor 2) to promote proliferation, survival, and growth of isolated neonatal and adult rat cardiac myocytes. Both ErbB2 and ErbB4 receptors were found to be expressed by neonatal and adult ventricular myocytes and activated by rhGGF2. rhGGF2 (30 ng/ml) provoked an approximate 2-fold increase in embryonic cardiac myocyte proliferation. rhGGF2 also promoted survival and inhibited apoptosis of subconfluent, serum-deprived myocyte primary cultures and also induced hypertrophic growth in both neonatal and adult ventricular myocytes, which was accompanied by enhanced expression of prepro-atrial natriuretic factor and skeletal alpha-actin. Moreover, NRG1 mRNA could be detected in coronary microvascular endothelial cell primary cultures prepared from adult rat ventricular muscle. NRG1 expression in these cells was increased by endothelin-1, another locally acting cardiotropic peptide within the heart. The persistent expression of both a neuregulin and its cognate receptors in the postnatal and adult heart suggests a continuing role for neuregulins in the myocardial adaption to physiologic stress or injury.

ARIA is heavily expressed in rat peripheral auditory and vestibular ganglia

The ARIA (acetylcholine receptor inducing activity) polypeptide is a member of the neuregulin gene family. It was originally purified on the basis of its ability to induce skeletal muscle nicotinic acetylcholine receptors (nAChRs). ARIA mRNA is expressed in ventral horn motor neurons and brain cholinergic neurons. We report here that ARIA mRNA is heavily expressed in the embryonic, developing, and adult peripheral auditory and vestibular ganglia, the spiral ganglion and Scarpa's ganglion. Neither ganglion is cholinergic, but both express mRNAs for nicotinic and muscarinic receptors. The expression of ARIA in these ganglia may be related to the regulation of cholinergic receptors or a more general role for ARIA in growth and development.

Maintenance of acetylcholine receptor number by neuregulins at the neuromuscular junction in vivo.

ARIA (for acetylcholine receptor-inducing activity), a protein purified on the basis of its ability to stimulate acetylcholine receptor (AChR) synthesis in cultured myotubes, is a member of the neuregulin family and is present at motor endplates. This suggests an important role for neuregulins in mediating the nerve-dependent accumulation of AChRs in the postsynaptic membrane. Nerve-muscle synapses have now been analyzed in neuregulin-deficient animals. Mice that are heterozygous for the deletion of neuregulin isoforms containing an immunoglobulin-like domain are myasthenic. Postsynaptic AChR density is significantly reduced, as judged by the decrease in the mean amplitude of spontaneous miniature endplate potentials and bungarotoxin binding. On the other hand, the mean amplitude of evoked endplate potentials was not decreased, due to an increase in the number of quanta released per impulse, a compensation that has been observed in other myasthenic states. Thus, the density of AChRs in the postsynaptic membrane depends on immunoglobulin-containing neuregulin isoforms throughout the life of the animal.

Axonal neuregulin signals cells of the oligodendrocyte lineage through activation of HER4 and Schwann cells through HER2 and HER3.

We are interested in the signaling between axons and glia that leads to myelination and maintenance of the myelin internode, and we have focused on the role of neuregulins and their receptors. Neuregulins are a family of ligands that includes heregulin, neu differentiation factor, glial growth factor, and the acetylcholine receptor-inducing activity. Three signal transducing transmembrane receptors for neuregulins, which bear significant homology to the EGF receptor, are currently known: HER2 (erbB2), HER3 (erbB3), and HER4 (erbB4). We have found that oligodendrocite-type II astrocyte (O2A) progenitor cells and mature oligodendrocytes express HER2 and HER4 but no HER3. Schwann cells express HER2 and HER3 but little HER4. In O2A progenitor cells and oligodendrocytes, recombinant neuregulin induces the rapid tyrosine phosphorylation of only HER4. HER2 is not phosphorylated in cells of the oligodendrocyte lineage, but a physical interaction between HER2 and HER4 was detected in coimmunoprecipitation experiments. In Schwann cells, neuregulin induces the phosphorylation of both HER2 and HER3. Coimmunoprecipitation experiments indicate that receptor activation in Schwann cells results in the formation of HER2:HER3 heterodimers. Neuregulin localized immunocytochemically was present on neurites of cultured dorsal root ganglion neurons, and it was released into the medium in a form that promoted receptor tyrosine phosphorylation. Neuregulins therefore meet important criteria expected of molecules involved in axonal-glial signaling. The use of unique neuregulin receptor combinations in oligodendrocytes and Schwann cells likely results in recruitment of different signaling pathways and thus provides a basis for different biological responses.

Identification of an Element Required for Acetylcholine Receptor-inducing Activity (ARIA)-induced Expression of the Acetylcholine Receptor ε Subunit Gene

Acetylcholine Receptor (AChR)-inducing activity (ARIA) is believed to be the trophic factor utilized by motoneurons to stimulate AChR synthesis in the subsynaptic area. Among the four AChR subunit genes, the epsilon subunit gene is strictly expressed in nuclei localized to the synaptic region of the muscle. To understand mechanisms of the regulation of synapse-specific transcription, we studied the promoter activity of the 5'-flanking region of the AChR epsilon subunit gene in response to ARIA. Transgenes containing the wild type or mutant 5'-flanking regions upstream of a luciferase gene were transfected in C2C12 muscle cells. The promoter activity of these transgenes was determined by assaying activity of expressed luciferase. Analyzing a combination of 5' deletion and site-directed mutants, we identified a 10-nucleotide element (position -55/-46), which was crucial for ARIA-induced expression from the epsilon subunit promoter. This element was named ARE for ARIA-responsive element. Mutation of ARE greatly diminished ARIA-induced transgene expression and deletion of ARE abolished completely the ARIA response. Electrophoretic mobility shift analyses revealed a DNA binding activity in muscle nuclear extract that interacted with ARE. Such interaction was enhanced by ARIA stimulation of muscle cells and appeared to be dependent on nuclear protein phosphorylation.

The EGF-like domain of chick acetylcholine receptor-inducing activity (ARIA) contains its full biological activity

Acetylcholine receptor-inducing activity (ARIA) is a glycoprotein initially purified from chick brain based on its ability to increase the synthesis of acetylcholine receptor (AChR) on cultured myotubes. cDNA encoding ARIA contains different domains and the functions of each domain in ARIA activity are not known. We used molecular genetic methods to construct a chimeric fusion protein, designated ARIA S136–K205-Fc, that contained the leader sequence, the EGF-like domain of chick ARIA (S 136 to K 205) and the Fc region of human immunoglobulin. The ARIA S136–K205-Fc cDNA was transfected into HEK 293 cells and stable cell lines secreting soluble ARIA S136–K205-Fc were obtained. The secreted ARIA S136–K205-Fc has a molecular mass of ∼60 kDa and can be purified by protein G chromatography. The purified ARIA S136–K205-Fc retained its full biological activity of chick ARIA that included: (i) induction of tyrosine phosphorylation of erbB 3 receptor in C2C12 myotubes; and (ii) ∼12-fold stimulation of AChR α-subunit mRNA synthesis when applied onto cultured chick myotubes. This Fc-tagged ARIA could be rapidly purified and provides a very useful ligand for identifying its true receptor(s) on muscle cell surface. © 1997 Federation of European Biochemical Societies.

Chick Muscle Expresses Various ARIA Isoforms: Regulation during Development, Denervation, and Regeneration

Acetylcholine receptor inducing activity (ARIA) is a glycoprotein released from the motor neuron to stimulate the synthesis of acetylcholine receptors (AChRs) on the postsynaptic muscle fiber. Transcripts encoding ARIA were detected not only in brain but also in muscle, and immunohistochemical staining showed that muscle-derived ARIA was restricted to the neuromuscular junctions. RT-PCR analysis revealed three biological active isoforms of ARIA in chick muscle, namely ARIAβ1, ARIAα2, and ARIAβ2that were classified based on their variation in the carboxyl-terminus of the EGF-like domain. The expression of these ARIA isoforms in muscle changed during development, denervation, and nerve regeneration. ARIAβ1, ARIAα2, and ARIAβ2were expressed in embryonic and young chick muscles, while ARIAβ1was the major isoform expressed in adult chicken. The embryonic-like expression of ARIAα2and ARIAβ2was induced after nerve injury in adult chicken. However, the prominent expression of ARIAβ1in adult-like profile was restored after nerve regeneration. A splicing variation in the region between Ig-like and EGF-like domains of ARIA was also revealed; a zero-amino acid insertion (ARIASP0), a 17-amino acid insertion (ARIASP17), or a 34-amino acid insertion (ARIASP34) were identified. Unlike ARIASP0, the expression of ARIASP17and ARIASP34was found in muscle and sciatic nerve only. The expression of ARIASP0, ARIASP17, and ARIASP34in chick muscle remained unchanged during development and after nerve injury. Moreover, the specific expression of these ARIA isoforms in cultured myotubes was not affected by drug treatments or by coculturing with neurons. Our findings provide strong evidence that muscle ARIA may play an important role in the formation of neuromuscular junctions.

Induction of Acetylcholine Receptor Gene Expression by ARIA Requires Activation of Mitogen-activated Protein Kinase

Transcription of genes encoding nicotinic acetylcholine receptor (AChR) subunits (alpha, beta, gamma or epsilon, and delta) is highest in nuclei localized to the synaptic region of the muscle, which contributes to maintain a high density of AChRs at the postjunctional membrane. ARIA (AChR inducing activity) is believed to be the trophic factor utilized by motor neurons to stimulate AChR synthesis in the subsynaptic area. To elucidate the signaling mechanism initiated by ARIA, we established stable C2C12 cell lines carrying the nuclear lacZ gene under the control of the mouse epsilon subunit promoter or chicken alpha subunit promoter. ARIA stimulated tyrosine phosphorylation of erbB proteins in these C2C12 cells within 15 s with a peak at 5 min. Immediately following tyrosine phosphorylation of erbB proteins, mitogen-activated protein (MAP) kinase was activated which occurred within 30 s and peaked at 8 min after ARIA stimulation. Concomitantly, expression of AChR genes was induced by ARIA. ARIA-induced AChR subunit transgene expression was observed only in differentiated myotubes and not in myoblasts, suggesting that downstream signaling component(s) are regulated in a manner dependent on the myogenic program. Inhibition of the MAP kinase activity by using a specific MAP kinase kinase inhibitor or by overexpressing dominant negative mutants of Raf or MAP kinase kinase attenuated or abolished the ARIA-induced activation of AChR alpha and epsilon subunit gene expression. These results indicate that regulation of AChR gene expression by ARIA in C2C12 cells requires activation of the MAP kinase signaling pathway.

Glial growth factors I-III are specific mitogens for glial cells.

Recently we identified three novel Schwann cell mitogens named GGF (glial growth factor)-I (34 kDa), GGF-II (59 kDa), and GGF-III (45 kDa), and provided evidence that they are three distinct but structurally related members of a larger family of factors, which includes heregulin, neu differentiation factor, and acetylcholine receptor-inducing activity (ARIA). We report here the characterization of the mitogenic and trophic activities for all three forms of GGF on rat Schwann cells and several other cell types. GGF-I, GGF-II, and GGF-III are potent mitogens for rat Schwann cells in vitro at nanomolar concentrations, whereas at lower concentrations they promote Schwann cell survival, in the absence of cAMP elevating agents. Forskolin, an adenylate cyclase activator, potently synergizes with the GGFs by an indirect mechanism, possibly involving transcriptional activation of GGF receptor(s). In addition, the GGFs stimulate DNA synthesis in rat glioma C6 cells, and in SK-BR-3 cells, which overexpress the p185 neu/erbB2. Fibroblasts obtained from different sources are weakly stimulated by GGFs, whereas PC12 cells are unable to respond under a variety of experimental conditions. These observations are consistent with the proposal that GGF-I, GGF-II, and GGF-III are a set of potent glial cell mitogens and putative ligands of members of the EGF receptor family, namely p185 neu/erbB2, p160/erbB3, and p180/erbB4, which may play important roles in the development, regeneration, and tumor biology of the peripheral nervous system.

Truncated form of pro-acetycholine receptor-inducing activity (ARIA) induces AChR α-subunit but not AChE transcripts in cultured chick myotubes

Acetylcholine receptor-inducing activity (ARIA) is a glycoprotein initially purified from chick brain based on its ability to increase the synthesis of acetylcholine receptor (AChR). We used reverse transcription-polymerase chain reaction (RT PCR) to obtain a partial pro-ARIA cDNA clone from methonine-1 to serine-358 including the full functional sequence of ARIA. Northern blot analysis of mRNAs from the embryonic chick brain and muscle showed a transcript with a size of ∼7.5 kb. The cloned cDNA was subcloned into an eukaryotic expression vector and stably transfected into human embryonic kidney 293 cells. The conditioned medium of the transfected cells was found to increase the level of transcript encoding for the α-subunit of AChR by ∼4.4-fold, but not for acetylcholinesterase (ACNE), in the cultured chick myotubes.

Neu differentiation factor is a neuron-glia signal and regulates survival, proliferation, and maturation of rat schwann cell precursors

We show that β forms of Neu differentiation factor (NDF), homologous to acetylcholine receptor-inducing activity, glial growth factor, and heregulin, prevent apoptotic death and stimulate DNA synthesis of the E14 Schwann cell precursor, an early cell in the rat Schwann cell lineage. When precursors are exposed to NDF in defined medium, they generate Schwann cells without the requirement for DNA synthesis and with a time course that is similar to that with which Schwann cells appear in embryonic nerves in vivo. Furthermore, a neuronal signal that also mediates precursor survival and maturation is blocked by the extracellular domain of the ErbB4 NDF receptor, a protein that specifically blocks the action of NDFs. These observations provide important evidence that NDF is one of the hitherto elusive neuron-glia signaling molecules long proposed to regulate development in the Schwann cell lineage.