Mendell Rimer

Agrin-Induced Postsynaptic-like Apparatus in Skeletal Muscle Fibersin Vivo

We find that when extrajunctional regions of denervated soleus muscles in adult rats are transfected with cDNA encoding rat agrin isoform Y4Z8, which is normally secreted by motor neurons at adult neuromuscular junctions, the myofibers express and secrete the neural agrin. Muscle fibers in the vicinity of transfection form at their surface specialized areas having extracellular, plasma membrane, and cytoplasmic protein aggregates, narrow and deep plasma membrane infoldings, and an accumulation of myonuclei, all of which are characteristic of the postsynaptic apparatus at neuromuscular junctions. We conclude that at ectopic neuromuscular junctions that form in the extrajunctional region of denervated adult soleus muscles after implantation of a foreign nerve, a single neural-derived factor, agrin, is sufficient not only to cause protein aggregation in the early stages of postsynaptic apparatus formation, as predicted by the agrin hypothesis, but also to bring about changes in conformation of the muscle fiber surface and distribution of organelles which appear as the apparatus reaches maturity.

Neuregulin-1 immunoglobulin-like domain mutant mice: clozapine sensitivity and impaired latent inhibition

Genetic and behavioral studies in humans and mouse mutants have implicated the gene encoding neuregulin-1 (Nrg-1) as a candidate susceptibility gene for schizophrenia. We examined the behavior of mice heterozygous for a mutation in neuregulin-1s immunoglobulin (Ig)-like domain (Ig-nrg-1+/− mice). We found that these animals displayed behaviors related to a schizophrenia-like phenotype, such as clozapine suppression of open-field and running wheel activity and impaired latent inhibition. Contrary to findings with other nrg-1 mutants, Ig-nrg-1+/− mice did not exhibit significantly elevated locomotion relative to littermate controls. These results suggest that Ig-Nrg-1s contribute to some – but not all – aspects of the schizophrenia-like phenotype of nrg-1 mutants, and further support nrg-1 as a candidate gene for schizophrenia.

Induction of Neuregulin Signaling in Mouse Schwann Cells In Vivo Mimics Responses to Denervation

Neuregulins play crucial roles in early development of Schwann cells (SCs), but their roles in the activities of SCs during denervation and reinnervation of muscle are less clear. In the present study, the Tet-On system has been used in transgenic mice to enable inducible expression of a mutant, constitutively active neuregulin receptor (ErbB2) in SCs. This induction simulates neuregulin signaling to these cells. Reporter transgenes were used to show a tightly regulated, SC-selective expression in muscle. Induction leads to a number of changes in SCs at neuromuscular junctions that mimic the response to muscle denervation/reinnervation. These include process extension, soma migration, and proliferation. SCs also come to express nestin, a protein characteristic of their reaction to muscle denervation. This activation of SCs results in the sprouting of nerve terminals, and these sprouts follow the extensions of the SCs. However, these sprouts and their associated SCs disappear after the removal of the inducer. Last, induction of the active receptor is sufficient to rescue SCs in neonatal muscle from denervation-induced apoptosis. These findings show that the responses of SCs in muscle to denervation can be explained by induction of an autocrine/paracrine neuregulin signaling cascade suggested by previous molecular studies.

γ-AChR/ϵ-AChR Switch at Agrin-Induced Postsynaptic-like Apparatus in Skeletal Muscle

Abstract We transfected the extrajunctional region of denervated soleus muscles in adult rats with neural agrin cDNA to induce myofibers to form postsynaptic-like apparatus containing acetylcholine receptor (AChR) aggregates. By 1 week ≈30% of the AChR aggregates contained a mixture of ϵ-AChRs and γ-AChRs while ≈70% had only γ-AChRs. If the transfected muscles were reinnervated in the original junctional region, the postsynaptic-like apparatus, despite the absence of apposed axon terminals, gradually came to have only ϵ-AChRs. We conclude that at the postsynaptic apparatus of ectopic neuromuscular junctions formed by a foreign nerve implanted into the extrajunctional region of denervated muscles, agrin secreted by the axon terminal plays a direct role in the γ-AChR/ϵ-AChR switch that occurs as the apparatus reaches maturity. Our findings, together with results from other studies, indicate further that agrin and acetylcholine are the only nerve-derived factors required for this switch.

Regulation of the Intermediate Filament Protein Nestin at Rodent Neuromuscular Junctions by Innervation and Activity

The intermediate filament nestin is localized postsynaptically at rodent neuromuscular junctions. The protein forms a filamentous network beneath and between the synaptic gutters, surrounds myofiber nuclei, and is associated with Z-discs adjacent to the junction. In situ hybridization shows that nestin mRNA is synthesized selectively by synaptic myonuclei. Although weak immunoreactivity is present in myelinating Schwann cells that wrap the preterminal axon, nestin is not detected in the terminal Schwann cells (tSCs) that cover the nerve terminal branches. However, after denervation of muscle, nestin is upregulated in tSCs and in SCs within the nerve distal to the lesion site. In contrast, immunoreactivity is strongly downregulated in the muscle fiber. Transgenic mice in which the nestin neural enhancer drives expression of a green fluorescent protein (GFP) reporter show that the regulation in SCs is transcriptional. However, the postsynaptic expression occurs through enhancer elements distinct from those responsible for regulation in SCs. Application of botulinum toxin shows that the upregulation in tSCs and the loss of immunoreactivity in muscle fibers occurs with blockade of transmitter release. Extrinsic stimulation of denervated muscle maintains the postsynaptic expression of nestin but does not affect the upregulation in SCs. Thus, a nestin-containing cytoskeleton is promoted in the postsynaptic muscle fiber by nerve-evoked muscle activity but suppressed in tSCs by transmitter release. Nestin antibodies and GFP driven by nestin promoter elements serve as excellent markers for the reactive state of SCs. Vital imaging of GFP shows that SCs grow a dynamic set of processes after denervation.

Neuregulin-2 is synthesized by motor neurons and terminal Schwann cells and activates acetylcholine receptor transcription in muscle cells expressing ErbB4.

Acetylcholine receptor (AChR) genes are transcribed selectively in synaptic nuclei of skeletal muscle fibers, leading to accumulation of the mRNAs encoding AChR subunits at synaptic sites. The signals that regulate synapse-specific transcription remain elusive, though Neuregulin-1 is considered a favored candidate. Here, we show that motor neurons and terminal Schwann cells express neuregulin-2, a neuregulin-1-related gene. In skeletal muscle, Neuregulin-2 protein is concentrated at synaptic sites, where it accumulates adjacent to terminal Schwann cells. Neuregulin-2 stimulates AChR transcription in cultured myotubes expressing ErbB4, as well as ErbB3 and ErbB2, but not in myotubes expressing only ErbB3 and ErbB2. Thus, Neuregulin-2 is a candidate for a signal that regulates synaptic differentiation.

Improvement of neuromuscular synaptic phenotypes without enhanced survival and motor function in severe spinal muscular atrophy mice selectively rescued in motor neurons.

In the inherited childhood neuromuscular disease spinal muscular atrophy (SMA), lower motor neuron death and severe muscle weakness result from the reduction of the ubiquitously expressed protein survival of motor neuron (SMN). Although SMA mice recapitulate many features of the human disease, it has remained unclear if their short lifespan and motor weakness are primarily due to cell-autonomous defects in motor neurons. Using Hb9Cre as a driver, we selectively raised SMN expression in motor neurons in conditional SMAΔ7 mice. Unlike a previous study that used choline acetyltransferase (ChATCre+) as a driver on the same mice, and another report that used Hb9Cre as a driver on a different line of conditional SMA mice, we found no improvement in survival, weight, motor behavior and presynaptic neurofilament accumulation. However, like in ChATCre+ mice, we detected rescue of endplate size and mitigation of neuromuscular junction (NMJ) denervation status. The rescue of endplate size occurred in the absence of an increase in myofiber size, suggesting endplate size is determined by the motor neuron in these animals. Real time-PCR showed that the expression of spinal cord SMN transcript was sharply reduced in Hb9Cre+ SMA mice relative to ChATCre+ SMA mice. This suggests that our lack of overall phenotypic improvement is most likely due to an unexpectedly poor recombination efficiency driven by Hb9Cre. Nonetheless, the low levels of SMN were sufficient to rescue two NMJ structural parameters indicating that these motor neuron cell autonomous phenotypes are very sensitive to changes in motoneuronal SMN levels. Our results directly suggest that even those therapeutic interventions with very modest effects in raising SMN in motor neurons may provide mitigation of neuromuscular phenotypes in SMA patients.

Neuregulins at the neuromuscular synapse: Past, present, and future

At the developing vertebrate neuromuscular junction, neuregulins are growth/differentiation factors essential for terminal Schwann cell survival. Neuregulins have also been thought as the critical signals responsible for the increased transcription of acetylcholine receptor subunit genes at the neuromuscular synapse. This latter role is now highly controversial. This article reviews the evidence that has shaped the views of the neuregulins and how these views have been challenged. The most recent experiments indicate that neuregulin signaling to postsynaptic muscle fibers may modulate, rather than determine, acetylcholine receptor expression at the neuromuscular junction. Based on findings from my lab and those of others, I propose that this modulation might involve novel posttranscriptional molecular mechanisms. Finally, I also suggest that neuregulin signaling may have an important role to play in mediating the response of adult terminal Schwann cells to denervation.

Neuregulins: Primary or secondary signals for the control of synapse-specific gene expression

The selective transcription of acetylcholine receptor (AChR) subunit genes in synaptic myonuclei leads to the accumulation of AChR subunit mRNAs at the neuromuscular junction (NMJ). This mechanism contributes to the concentration of AChRs at the postsynaptic sarcolemma, and its physiological significance is underscored by the cases of human congenital myasthenias caused by mutation in a cis-regulatory element of the AChRε-subunit promoter, which is necessary for its synaptic expression. The signal(s) that drives synapse-specific expression is unknown but neuregulin-1 (Nrg-1), a group of growth-factor-like polypeptides encoded by the nrg-1 gene, has been a favorite candidate. Nrg-1 was originally thought as a nerve-derived factor, acting in parallel to pathways controlling AChR clustering at the synapse (i.e. agrin signaling). However, recent work suggests that Nrg-1 may actually be a muscle-derived signal that is concentrated at the NMJ, together with its receptors, by agrin and that acts as a secondary, downstream signal to enhance synapse-specific AChR transcription. Here, I review studies for and against Nrg-1 as a secondary signal driving synapse-specific expression at the NMJ. In addition, I briefly present new evidence that raise the possibility that Nrgs encoded by the ngr-1-related gene nrg-2 might have a role controlling AChR expression.

Defective neuromuscular synaptogenesis in mice expressing constitutively active ErbB2 in skeletal muscle fibers

We overexpressed a constitutively active form of the neuregulin receptor ErbB2 (CAErbB2) in skeletal muscle fibers in vivo and in vitro by tetracycline-inducible expression. Surprisingly, CAErbB2 expression during embryonic development was lethal and impaired synaptogenesis yielding a phenotype with loss of synaptic contacts, extensive axonal sprouting, and diffuse distribution of acetylcholine receptor (AChR) transcripts, reminiscent of agrin-deficient mice. CAErbB2 expression in cultured myotubes inhibited the formation and maintenance of agrin-induced AChR clusters, suggesting a muscle- and not a nerve-origin for the defect in CAErbB2-expressing mice. Levels of tyrosine phosphorylated MuSK, the signaling component of the agrin receptor, were similar, while tyrosine phosphorylation of AChRbeta subunits was dramatically reduced in CAErbB2-expressing embryos relative to controls. Thus, a gain-of-function manipulation of ErbB2 signaling pathways renders an agrin-deficient-like phenotype that uncouples MuSK and AChR tyrosine phosphorylation.