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Biomedical subjects

J P Merlie

Publications and source records attributed to J P Merlie.

At least 19 recordsLinked to original sources

Failure of postsynaptic specialization to develop at neuromuscular junctions of rapsyn-deficient mice.

Of numerous synaptic components that have been identified, perhaps the best-studied are the nicotinic acetylcholine receptors (AChRs) of the vertebrate neuromuscular junction. AChRs are diffusely distributed on embryonic myotubes, but become highly concentrated (approximately 10,000 microns-2) in the postsynaptic membrane as development proceeds. At least two distinct processes contribute to this accumulation. One is local synthesis: subsynaptic muscle nuclei transcribe AChR subunit genes at higher rates than extra-synaptic nuclei, so AChR messenger RNA is concentrated near synaptic sites. Second, once AChRs have been inserted in the membrane, they form high-density clusters by tethering to a subsynaptic cytoskeletal complex. A key component of this complex is rapsyn, a peripheral membrane protein of relative molecular mass 43K (refs 4, 5), which is precisely colocalized with AChRs at synaptic sites from the earliest stages of neuromuscular synaptogenesis. In heterologous systems, expression of recombinant rapsyn leads to clustering of diffusely distributed AChRs, suggesting that rapsyn may control formation of clusters. To assess the role of rapsyn in vivo, we generated and characterized mutant mice with a targeted disruption of the Rapsyn gene. We report that rapsyn is essential for the formation of AChR clusters, but that synapse-specific transcription of AChR subunit genes can proceed in its absence.

Animals

Aberrant differentiation of neuromuscular junctions in mice lacking s-laminin/laminin beta 2.

Synapse formation requires a complex interchange of information between the pre- and postsynaptic partners. At the skeletal neuromuscular junction, some of this information is contained in the basal lamina (BL), which runs through the synaptic cleft between the motor nerve terminal and the muscle fibre. During regeneration following injury, components of synaptic BL can trigger several features of postsynaptic differentiation in the absence of the nerve terminal, and of presynaptic differentiation in the absence of the muscle fibre. One nerve-derived component of synaptic BL, agrin, is known to affect postsynaptic differentiation, but no muscle-derived components have yet been shown to influence motor nerve terminals. A candidate for such a role is s-laminin (also called laminin beta 2), a homologue of the B1 (beta 1) chain of the widely distributed BL glycoprotein, laminin. s-Laminin is synthesized by muscle cells and concentrated in synaptic BL. In vitro, recombinant s-laminin fragments are selectively adhesive for motor neuron-like cells, inhibit neurite outgrowth promoted by other matrix molecules, and act as a 'stop signal' for growing neurites. By generating and characterizing mice with a targeted mutation of the s-laminin gene, we show here that s-laminin regulates formation of motor nerve terminals.

Action Potentials

Myasthenia gravis-like syndrome induced by expression of interferon gamma in the neuromuscular junction.

Abnormal humoral responses toward motor end plate constituents in muscle induce myasthenia gravis (MG). To study the etiology of this disease, and whether it could be induced by host defense molecules, we examined the consequences of interferon (IFN) gamma production within the neuromuscular junction of transgenic mice. The transgenic mice exhibited gradually increasing muscular weakness, flaccid paralysis, and functional disruption of the neuromuscular junction that was reversed after administration of an inhibitor of acetylcholinesterase, features which are strikingly similar to human MG. Furthermore, histological examination revealed infiltration of mononuclear cells and autoantibody deposition at motor end plates. Immunoprecipitation analysis indicated that a previously unidentified 87-kD target antigen was recognized by sera from transgenic mice and also by sera from the majority of human MG patients studied. These results suggest that expression of IFN-gamma at motor end plates provokes an autoimmune humoral response, similar to human MG, thus linking the expression of this factor with development of this disease.

Animals

Activation of the myogenin promoter during mouse embryogenesis in the absence of positive autoregulation.

Myogenin, a member of the MyoD family of helix-loop-helix proteins, can induce myogenesis in a wide range of cell types. In addition to activating muscle structural genes, members of the MyoD family can autoactivate their own and cross-activate one another's expression in transfected cells. This has led to the hypothesis that autoregulatory loops among these factors provide a mechanism for amplifying and maintaining the muscle-specific gene expression program in vivo. Here, we make use of myogenin-null mice to directly test this hypothesis. To investigate whether the myogenin protein autoregulates the myogenin gene during embryogenesis, we introduced a myogenin-lacZ transgene into mice harboring a null mutation at the myogenin locus. Despite a severe deficiency of skeletal muscle in myogenin-null neonates, the myogenin-lacZ transgene was expressed normally in myogenic cells throughout embryogenesis. These results show that myogenin is not required for regulation of the myogenin gene and argue against the existence of a myogenin autoregulatory loop in the embryo.

Animals

Synapse-associated expression of an acetylcholine receptor-inducing protein, ARIA/heregulin, and its putative receptors, ErbB2 and ErbB3, in developing mammalian muscle.

Developing motor axons induce synaptic specializations in muscle fibers, including preferential transcription of acetylcholine receptor (AChR) subunit genes by subsynaptic nuclei. One candidate nerve-derived signaling molecule is AChR-inducing activity (ARIA)/heregulin, a ligand of the erbB family of receptor tyrosine kinases. Here, we asked whether ARIA and erbB kinases are expressed in patterns compatible with their proposed signaling roles. In developing muscle, ARIA was present not only at synaptic sites, but also in extrasynaptic regions of the muscle fiber. ARIA was synthesized, rather than merely taken up, by muscle cells, as indicated by the presence of ARIA mRNA in muscle and of ARIA protein in a clonal muscle cell line. ARIA-responsive myotubes expressed both erbB2 and erbB3, but little EGFR/erbB1 or erbB4. In adults, erbB2 and erbB3 were localized to the postsynaptic membrane. ErbB3 was restricted to the postsynaptic membrane perinatally, at a time when ARIA was still broadly distributed. Thus, our data are consistent with a model in which ARIA interacts with erbB kinases on the muscle cell surface to provide a local signal that induces synaptic expression of AChR genes. However, much of the ARIA is produced by muscle, not nerve, and the spatially restricted response may result from the localization of erbB kinases as well as of ARIA. Finally, we show that erbB3 is not concentrated at synaptic sites in mutant mice that lack rapsyn, a cytoskeletal protein required for AChR clustering, suggesting that pathways for synaptic AChR expression and clustering interact.

Aging

N-CAM, 43K-rapsyn, and S-laminin mRNAs are concentrated at synaptic sites in muscle fibers.

Several components of the postsynaptic apparatus are found highly concentrated at the motor endplate. Studies of the acetylcholine receptor have shown that selective transcription of its genes by synaptic nuclei contributes to its synaptic accumulation. We used the method of in situ hybridization to study the distribution of mRNAs encoding three other proteins localized to the motor endplate. We found preferential synaptic accumulation of mRNAs for a membrane-associated cell adhesion molecule (N-CAM) and for an acetylcholine receptor-associated cytoskeletal protein (43K-rapsyn). In contrast, RNAs encoding proteins present throughout the muscle were distributed all along the muscle fiber. RNA encoding a protein concentrated in synaptic basal lamina, s-laminin (laminin beta 2), was intermediate in distribution, detectable extrasynaptically but more abundant synaptically. Our data suggest that selective transcription by synaptic nuclei is a general mechanism that contributes to the concentration of specific proteins in the postsynaptic apparatus at the neuromuscular junction.

Animals

Rapsyn may function as a link between the acetylcholine receptor and the agrin-binding dystrophin-associated glycoprotein complex.

The 43 kDa AChR-associated protein rapsyn is required for the clustering of nicotinic acetylcholine receptors (AChRs) at the developing neuromuscular junction, but the functions of other postsynaptic proteins colocalized with the AChR are less clear. Here we use a fibroblast expression system to investigate the role of the dystrophin-glycoprotein complex (DGC) in AChR clustering. The agrin-binding component of the DGC, dystroglycan, is found evenly distributed across the cell surface when expressed in fibroblasts. However, dystroglycan colocalizes with AChR-rapsyn clusters when these proteins are coexpressed. Furthermore, dystroglycan colocalizes with rapsyn clusters even in the absence of AChR, indicating that rapsyn can cluster dystroglycan and AChR independently. Immunofluorescence staining using a polyclonal antibody to utrophin reveals a lack of staining of clusters, suggesting that the immunoreactive species, like the AChR, does not mediate the observed rapsyndystroglycan interaction. Rapsyn may therefore be a molecular link connecting the AChR to the DGC. At the neuromuscular synapse, rapsyn-mediated linkage of the AChR to the cytoskeleton-anchored DGC may underlie AChR cluster stabilization.

Agrin

Regulation of the acetylcholine receptor epsilon subunit gene by recombinant ARIA: an in vitro model for transynaptic gene regulation.

Structural specialization of the postsynaptic skeletal muscle membrane is in part mediated by the motor neuron-induced transcriptional regulation of synaptic muscle nuclei. ARIA, a factor that stimulates production of acetylcholine receptors (AChRs), is a candidate signaling molecule for such regulation. Here we examine the transynaptic inducing potential of this polypeptide factor. ARIA immunoreactivity is detectable at synaptic sites in vivo. In vitro, recombinant heregulin beta 1 (rHRG beta 1), the human homolog of ARIA, induces expression of the AChR epsilon gene, the subunit most sensitive to synaptic input. The inducing property of rHRG beta 1 is demonstrated most dramatically in primary muscle cultures from transgenic mice bearing an epsilon promoter-nuclear lacZ reporter transgene. Transient transfection experiments using the Sol 8 muscle cell line indicate that sequences that confer responsiveness to ARIA are located within a 150 bp epsilon subunit promoter region and are E box-independent. These results suggest that ARIA performs a vital role by directing spatially restricted gene expression at the neuromuscular junction.

Animals

Assembly of the postsynaptic apparatus.

Recent research has led to a clearer picture of the molecular organization of the postsynaptic apparatus at the developing neuromuscular junction. In addition, one link between the extracellular signaling molecule agrin and the intracellular events that mediate formation of acetylcholine receptor clusters has been established with the identification of an argin-binding protein.

Agrin

The renal glomerulus of mice lacking s-laminin/laminin beta 2: nephrosis despite molecular compensation by laminin beta 1.

S-laminin/laminin beta 2, a homologue of the widely distributed laminin B1/beta 1 chain, is a major component of adult renal glomerular basement membrane (GBM). Immature GBM bears beta 1, which is replaced by beta 2 as development proceeds. In mutant mice that lack beta 2, the GBM remains rich in beta 1, suggesting that a feedback mechanism normally regulates GBM maturation. The beta 2-deficient GBM is structurally intact and contains normal complements of several collagenous and noncollagenous glycoproteins. However, mutant mice develop massive proteinuria due to failure of the glomerular filtration barrier. These results support the idea that laminin beta chains are functionally distinct although they assemble to form similar structures. Laminin beta 2-deficient mice may provide a model for human congenital or idiopathic nephrotic syndromes.

Animals

The 3' flanking region of the human tyrosine hydroxylase gene directs reporter gene expression in peripheral neuroendocrine tissues.

Cell type-specific expression of the catecholamine synthetic enzyme, tyrosine hydroxylase (TH), appears to be mediated in part by cis-acting elements located at the 3' end of the human gene. Further delineation of this region indicated sequences corresponding to a CACGTG motif significantly stimulated transcription of a heterologous promoter in various cell types. Mutation of this site led to a complete loss of activity. DNase footprinting, gel retardation, and UV cross-linking experiments indicated that a 74-kDa cellular factor(s) bound specifically to the CACGTG motif in the pheochromocytoma cell line PC12. The size of this protein and its pattern of expression are compatible with those of the CACGTG binding protein TFE3. Transgenic animals were created using a 261-bp human TH 3' fragment encompassing the CACGTG motif in front of a thymidine kinase promoter/chloramphenicol acetyltransferase reporter gene. In three lines of mice this fragment was sufficient to direct a pattern of mRNA expression in peripheral neuroendocrine tissues that mimicked TH mRNA distribution. However, these sequences were not sufficient for CNS-specific patterns of expression. Thus, multiple cell type-specific enhancers may regulate TH gene expression in the CNS and periphery.

Animals

Differential expression of voltage-gated K+ channel subunits in adult rat heart. Relation to functional K+ channels?

Polyclonal antibodies against each of the K+ channel subunits (Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2) shown previously to be expressed in adult rat heart at the mRNA level were used to examine the distributions of these K+ channel subunits in adult rat atrial and ventricular membranes. Immunohistochemistry on isolated adult rat ventricular myocytes revealed strong labeling with the anti-Kv4.2 and anti-Kv1.2 antibodies. Although somewhat weaker (than with anti-Kv1.2 or anti-Kv4.2), positive staining was also observed with the anti-Kv1.5 and anti-Kv2.1 antibodies. Ventricular myocytes exposed to the anti-Kv1.4 antibody, in contrast, did not appear significantly different from background. Qualitatively similar results were obtained on isolated adult rat atrial myocytes. Western blots of atrial and ventricular membrane proteins confirmed the presence of Kv1.2, Kv1.5, Kv2.1, and Kv4.2 and revealed differences in the relative abundances of these subunits in the two membrane preparations. Kv4.2, for example, is more abundant in ventricular than in atrial membranes, whereas Kv1.2 and Kv2.1 are higher in atrial membranes; Kv1.5 levels are comparable in the two preparations. In contrast to these results, nothing was detected in Western blots of atrial or ventricular membrane proteins with the anti-Kv1.4 antibody at concentrations that revealed intense labeling of a 97-kD protein in adult rat brain membranes. A very faint band was detected at 97 kD in the atrial and ventricular preparations when the anti-Kv1.4 antibody was used at a 5- to 10-fold higher concentration. The simplest interpretation of these results is that Kv1.4 is not an abundant protein in adult rat atrial or ventricular myocytes. Therefore, it seems unlikely that Kv1.4 plays an important role in the formation of functional depolarization-activated K+ channels in these cells. The relation(s) between the (other four) K+ channel subunits and the depolarization-activated K+ channels identified electrophysiologically in adult rat atrial and ventricular myocytes is discussed in the present study.

Animals

Characterization and mapping of the Rapsn gene encoding the 43-kDa acetylcholine receptor-associated protein.

We have cloned and characterized mouse genomic DNA containing the gene for the 43-kDa acetylcholine receptor-associated protein. The gene extends over 12 kb and consists of 8 exons. RNase protection and sequence analysis have been used to define the intron/exon boundaries including 174 and 214 bp of 5' and 3' untranslated sequence in exons 1 and 8, respectively. Interestingly, the exon/intron organization is consistent with structural domains predicted from amino acid sequence conservation among 3 species of 43K. Finally, the 43K locus, designated Rapsn, has been mapped to the central region of mouse chromosome 2.

Amino Acid Sequence

Id-1 as a possible transcriptional mediator of muscle disuse atrophy.

Disuse of muscle leads to atrophy of the fibers. This atrophy is correlated with reduced transcription. We found that when muscle was denervated or paralyzed with a nerve impulse block, the mRNA for Id-1, a negative regulator of transcription, was increased 2- to 7-fold. To test the effect of high Id-1 levels in active muscles, we made transgenic mice in which Id-1 was overexpressed under control of regulatory elements which confer tissue- and fiber-type-specific expression in differentiated muscle cells. Fiber types with high transgene expression were atrophic compared to those in wild-type litter mates. In contrast, fiber types with low transgene expression displayed hypertrophy, presumably caused by an overload due to reduced strength in atrophic synergistic fibers. Apart from the selective effects on fiber caliber, the muscle tissue showed no signs of pathology, and apart from a characteristic slightly lower body weight, the transgenic animals looked and behaved normally. We suggest that in the mature muscle, Id-1 may be involved in regulating muscle fiber size at the transcriptional level during disuse.

Animals

Interspecific comparisons reveal conserved features of the Drosophila Toll protein.

The Toll gene of Drosophila melanogaster produces a transmembrane cell adhesion protein that is required to establish the dorso-ventral axis of the embryo. The Toll protein's extracellular domain contains Leu-rich repeats (LRR), implicated in intermolecular interactions, and its large intracellular domain transduces a signal that eventually reaches the nucleus. Here, we report amino-acid (aa) sequences encoded by the Toll genes of D. pseudoobscura and D. virilis, and two distinct Toll-like genes of the grasshopper, Schistocerca americana. Interspecific comparisons show a Toll-specific subfamily of LRR, and a strikingly high degree of conservation in the cytoplasmic domain. Interestingly, many aa residues conserved among the insect Toll-like cytoplasmic domains are also conserved in mammalian and avian type-I interleukin-1 receptors and the hypothetical product of a transcript, MyD88, found in murine myeloid cells. Thus, we identify a set of conserved aa in the cytoplasmic domain which might be used in a signal-transduction pathway shared by invertebrates and vertebrates.

Amino Acid Sequence

Myogenin and acetylcholine receptor alpha gene promoters mediate transcriptional regulation in response to motor innervation.

Several genes expressed in skeletal muscle are transcriptionally repressed by electrical activity arising from motor innervation and are rapidly induced following denervation. Among these are genes encoding the subunits of the nicotinic acetylcholine receptor (AChR) and the myogenic helix-loop-helix protein myogenin, which activates muscle-specific genes. To understand how electrical activity arising from motor innervation is converted into a transcriptional response, we have attempted to localize cis-acting sequences in the AChR alpha subunit and myogenin genes sufficient to direct activity-dependent transcription. Here we show that an 111-base pair and a 335-base pair region from the promoters of the AChR alpha subunit and myogenin genes, respectively, can confer activity-dependent regulation to a linked reporter gene in transgenic mice. The presence of binding sites for myogenic helix-loop-helix proteins in both of these regulatory regions is consistent with the hypothesis that these myogenic regulators serve as nuclear targets for the signaling cascade through which motor innervation leads to changes in gene transcription in skeletal muscle.

Aging

A novel synapse-associated noncoding RNA.

Synaptic nuclei of innervated muscle transcribe acetylcholine receptor (AChR) genes at a much higher level than extrasynaptic nuclei. To isolate candidate synaptic regulatory molecules responsible for the unique transcriptional potential of synaptic nuclei, we have taken a subtractive hybridization approach. Here, we report the cloning and characterization of a novel synapse-associated RNA, 7H4. 7H4 is expressed selectively in the endplate zone of skeletal muscle and is upregulated during early postnatal development and after denervation. Interestingly, the 7H4 gene has no introns, and yet two different-size RNAs with identical polyadenylated 3' ends are generated. Most intriguingly, the nucleotide sequence does not contain any significant open reading frames, suggesting that 7H4 may function as a noncoding RNA.

Animals

Cell type- and differentiation-dependent expression from the mouse acetylcholine receptor epsilon-subunit promoter.

The nicotinic acetylcholine receptor (AChR) in adult skeletal muscle is composed of alpha-, beta-, epsilon-, and delta-subunits and is localized at the neuromuscular junction; in contrast, the more diffusely distributed fetal form is composed of alpha-, beta-, gamma-, and delta-subunits. To define sequences necessary for the transcriptional regulation of the mouse epsilon-subunit gene, we sequenced and analyzed 1036 bp upstream of the transcription start site. Using deletion analysis of the 5'-flanking region linked to the bacterial chloramphenicol acetyltransferase (CAT) gene and transfection of the resulting constructs into established cell lines, we demonstrate that a 151 bp fragment exhibits cell type- and differentiation-specific promoter activity. This activity was independent of a myogenic factor putative binding site (E-box). However, transactivation experiments with recombinant myoD, myogenin, or MRF4 showed that the E-box was functional and that MRF4 preferentially transactivates the epsilon-promoter. Thus, like other AChR promoters, the proximal region of the epsilon-promoter contains information for cell type-specific and developmental regulation of CAT and can be transactivated by myogenic factors in cultured cell lines. Unlike the other AChR promoters characterized to date, epsilon-promoter function can be partially independent of myogenic factors of the helix-loop-helix class.

Animals