Biomedical subjects

J P Merlie

Publications and source records attributed to J P Merlie.

At least 37 records · Page 2Linked to original sources

PubMed1995

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↗

PubMed1995

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↗

PubMed1995

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↗

PubMed1995

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↗

PubMed1995

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↗

PubMed1995

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↗

PubMed1995

Overexpression of myogenin in muscles of transgenic mice: interaction with Id-1, negative crossregulation of myogenic factors, and induction of extrasynaptic acetylcholine receptor expression.

To investigate the role of myogenin in regulating acetylcholine receptor expression in adult muscle, this muscle-specific basic helix-loop-helix transcription factor was overexpressed in transgenic mice by using regulatory elements conferring strong expression confined to differentiated postmitotic muscle fibers. Many of the transgenic mice died during the first postnatal week, but those that survived into adulthood displayed normal muscle histology, gross morphology, and motor behavior. The mRNA levels of all five acetylcholine receptor subunits (alpha, beta, gamma, delta, and epsilon) were, however, elevated. Also, the level of receptor protein was increased and high levels of receptors were present throughout the extrasynaptic surface membrane of the muscle fibers. Thus, elevated levels of myogenin are apparently sufficient to induce acetylcholine supersensitivity in normally innervated muscle of adult mice. The high neonatal mortality rate of the mice overexpressing myogenin hindered the propagation of a stable line. In an attempt to increase survival, myogenin overexpressers were mated with a line of transgenic mice overexpressing Id-1, a negative regulator that interacts with the basic helix-loop-helix family of transcription factors. The Id-1 transgene apparently worked as a second site suppressor and abolished the high rate of neonatal mortality. This effect indicates that Id-1 and myogenin interact directly or indirectly in these animals. Further study indicated that myogenin overexpression had no effect on the level of endogenous myogenin mRNA, while the levels of myoD and MRF4 mRNAs were reduced. Overexpression of the negative regulator Id-1 increased the mRNA levels of all the myogenic factors. These findings are consistent with a hypothesis suggesting that myogenic factors are influenced by mechanisms that maintain cellular homeostasis.

Aging↗

PubMed1995

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↗

PubMed1994

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↗

PubMed1994

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↗

PubMed1994

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↗

PubMed1994

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↗

PubMed1994

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↗

PubMed1993

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↗

PubMed1993

Separable regulatory elements governing myogenin transcription in mouse embryogenesis.

Expression of the myogenic helix-loop-helix (HLH) protein myogenin in muscle cell precursors within somites and limb buds is among the earliest events associated with myogenic lineage determination in vertebrates. Mutations in the myogenin promoter that abolish binding sites for myogenic HLH proteins or myocyte enhancer factor-2 (MEF-2) suppressed transcription of a linked lacZ transgene in subsets of myogenic precursors in mouse embryos. These results suggest that myogenic HLH proteins and MEF-2 participate in separable regulatory circuits leading to myogenin transcription and provide evidence for positional regulation of myogenic regulators in the embryo.

Animals↗

PubMed1993

43K protein and acetylcholine receptors colocalize during the initial stages of neuromuscular synapse formation in vivo.

The 43K protein is a cytoplasmic peripheral membrane protein concentrated subsynaptically in skeletal muscle. Recombinant 43K has been shown to cause clustering of acetylcholine receptors (AChRs) in cultured cells. However, the role of 43K in vivo is disputed, because in some cases it appears only after AChRs have clustered. We therefore examined the expression and distribution of 43K and AChRs during synapse formation in embryonic mouse muscles. Messenger RNA for 43K was detected on Embryonic Day (E) 12, a day prior to the first AChR clusters. Immunofluorescence showed that both AChRs and 43K were colocalized in patches by E13, the stage at which intramuscular nerves were first detected. The AChR/43K patches were nerve associated, and more than 98% of AChR patches were accompanied by 43K. The precise colocalization of 43K and AChRs persisted through development. These results are consistent with 43K being involved in the nerve-induced clustering of AChRs during synapse formation.

Animals↗

PubMed1993

S-laminin: mapping to mouse chromosome 9 and expression in the linked mutants tippy and ducky.

S-Laminin, a homologue of the laminin B1 chain, is present in a subset of basal laminae, including those of the skeletal neuromuscular junction and the renal glomerulus. Here, we show that the distribution and apparent size of murine S-laminin are similar to those documented previously for rat and human. We then use interspecific backcross analysis to map the S-laminin (Lams) gene to mouse chromosome 9. Thus, it is unlinked to genes for the laminin A, B1, and B2 chains. Finally, because the Lams gene mapped near two mutations that affect neuromuscular function, ducky (du) and tippy (tip), we assayed S-laminin by Southern blotting, immunoblotting, and immunohistochemistry in these mutants. No abnormality of the S-laminin gene or protein was detectable in either mutant.

Animals↗

PubMed1993

Interaction of the 43 kd postsynaptic protein with all subunits of the muscle nicotinic acetylcholine receptor.

The 43 kd postsynaptic protein (43K) plays a key role in the aggregation of muscle nicotinic acetylcholine receptors (AChRs) in the postsynaptic membrane of the neuromuscular junction. By transiently coexpressing 43K and a single AChR subunit (alpha, beta, gamma, or delta) in the quail fibroblast cell line, QT-6, we show that 43K interacts with each subunit to form cell surface clusters in which 43K and receptor subunit are precisely colocalized. Although the level of cell surface expression of single subunits is much lower than that of fully assembled receptor, the clustering of both single subunits and fully assembled AChR occurs efficiently. In addition, 43K-induced clustering is specific for AChR subunits. From these results, we conclude that each pentameric AChR has five potential sites for interacting with 43K during cluster formation.

Animals↗