Hans R. Brenner

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 AChRɛ . We propose that the novel pathway stabilizes the synapse early in development, whereas the NRG/ErbB pathway supports maintenance of the mature synapse.

Neurotrophic control of channel properties at neuromuscular synapses of rat muscle

1. Ectopic neuromuscular synapses formed when the fibular nerve was implanted into the proximal part of rat soleus muscle and the soleus nerve was cut. The gating properties of acetylcholine (ACh) receptor channels in the newly formed ectopic and in the denervated original end‐plates were examined at various stages of ectopic synapse formation.

Agrin regulates CLASP2-mediated capture of microtubules at the neuromuscular junction synaptic membrane

Agrin regulates acetylcholine receptors at the neuromuscular junction by locally stabilizing microtubules through the plus end tracking proteins CLASP2 and CLIP-170.

Gating properties of acetycholine receptor in newly formed neuromuscular synapses

A FOREIGN motor nerve transplanted on to an adult innervated muscle will not form functional neuromuscular synapses1, yet several experimental procedures, such as denervation or muscle crush, change the properties of the muscle membrane so that a foreign nerve can establish ‘ectopic’ synapses in a normally endplate-free region of the muscle2–4. The nerve thereby induces a localised area of high acetylcholine receptor (AChR) density in the extrajunctional membrane5. Denervated or previously crushed muscle fibres already have, before synapse formation, low density AChRs in the extrajunctional membrane6,7. The extrajunctional AChRs are different in a number of respects from those in the junctional membrane8–11. The question is, therefore, whether the AChRs of an ectopic synapse have properties similar to junctional or to extrajunctional AChRs.

A Novel Role for Embigin to Promote Sprouting of Motor Nerve Terminals at the Neuromuscular Junction

Adult skeletal muscle accepts ectopic innervation by foreign motor axons only after section of its own nerve, suggesting that the formation of new neuromuscular junctions is promoted by muscle denervation. With the aim to identify new proteins involved in neuromuscular junction formation we performed an mRNA differential display on innervated versus denervated adult rat muscles. We identified transcripts encoding embigin, a transmembrane protein of the immunoglobulin superfamily (IgSF) class of cell adhesion molecules to be strongly regulated by the state of innervation. In innervated muscle it is preferentially localized to neuromuscular junctions. Forced overexpression in innervated muscle of a full-length embigin transgene, but not of an embigin fragment lacking the intracellular domain, promotes nerve terminal sprouting and the formation of additional acetylcholine receptor clusters at synaptic sites without affecting terminal Schwann cell number or morphology, and it delays the retraction of terminal sprouts following re-innervation of denervated endplates. Conversely, knockdown of embigin by RNA interference in wild-type muscle accelerates terminal sprout retraction, both by itself and synergistically with deletion of neural cell adhesion molecule. These findings indicate that embigin enhances neural cell adhesion molecule-dependent neuromuscular adhesion and thereby modulates neuromuscular junction formation and plasticity.

Gene transfer into individual muscle fibers and conditional gene expression in living animals

Abstract. Pressure injection of DNA directly into individual fibers of surgically exposed soleus muscle leads to efficient and reliable expression of the transgene. Conditionally regulated gene expression in a single muscle fiber was analyzed in vivo by co-injecting a tetracycline-regulated lacZ reporter construct and a transactivator (rtTA) expression vector. The tetracycline-responsive element revealed significant basal transcriptional activity that was further increased by rtTA even in the absence of the effector doxycycline (dox). The high basal activity of the simple two-component system precludes tight gene regulation in muscle. Concomitant expression of the silencer tTSKid, however, reduced the basal activity to low or undetectable levels. This allowed the specific activation of the tetracycline-responsive element by the application of dox. Direct gene transfer can thus be employed to express transgenic proteins in distinct muscle fibers at spatially defined regions and to regulate gene expression conditionally.