Hans Rudolf Brenner

Acetylcholine receptor α-, β-, γ-, and δ-subunit mRNA levels are regulated by muscle activity

Abstract Denervation of adult skeletal muscle results in increased sensitivity to acetylcholine in extrajunctional regions of the muscle fiber. This increase in acetylcholine sensitivity is accompanied by a large increase in the level of mRNAs coding for the α-, β-, γ-, and δ-subunits of the acetylcholine receptor. To determine whether muscle activity is sufficient to regulate expression of extrajunctional acetylcholine receptor mRNA levels, denervated muscles were stimulated with extracellular electrodes. Direct stimulation of denervated muscle suppresses both the increase in extrajunctional acetylcholine sensitivity and the expression of mRNA encoding the α-, β-, γ-, and δ-subunits of the acetylcholine receptor. These results show that muscle activity regulates the level of extrajunctional acetylcholine receptors by regulating the expression of their mRNAs.

Erbb2 regulates neuromuscular synapse formation and is essential for muscle spindle development.

Neuregulins and their Erbb receptors have been implicated in neuromuscular synapse formation by regulating gene expression in subsynaptic nuclei. To analyze the function of Erbb2 in this process, we have inactivated the Erbb2 gene in developing muscle fibers by Cre/Lox-mediated gene ablation. Neuromuscular synapses form in the mutant mice, but the synapses are less efficient and contain reduced levels of acetylcholine receptors. Surprisingly, the mutant mice also show proprioceptive defects caused by abnormal muscle spindle development. Sensory Ia afferent neurons establish initial contact with Erbb2-deficient myotubes. However, functional spindles never develop. Taken together, our data suggest that Erbb2 signaling regulates the formation of both neuromuscular synapses and muscle spindles.

Neuregulin/ErbB regulate neuromuscular junction development by phosphorylation of α-dystrobrevin

Neuregulin/ErbB signaling maintains high efficacy of synaptic transmission by stabilizing the postsynaptic apparatus via phosphorylation of α-dystrobrevin1.

The role of nerve- versus muscle-derived factors in mammalian neuromuscular junction formation

Neuromuscular junctions (NMJs) normally form in the central region of developing muscle. In this process, agrin released from motor neurons has been considered to initiate the formation of synaptic acetylcholine receptor (AChR) clusters (neurocentric model). However, in muscle developing in the absence of nerves and thus of agrin, AChR clusters still form in the muscle center. This raises the possibility that the region of NMJ formation is determined by muscle-derived cues that spatially restrict the nerve to form synapses from aneural AChR clusters, e.g., by patterned expression of the agrin receptor MuSK (muscle-specific kinase) (myocentric model). Here we examine at initial stages of synaptogenesis whether the responsiveness of myotubes to agrin is spatially restricted, whether the regions of NMJ formation in wild-type muscle and of aneural AChR cluster formation in agrin-deficient animals correlate, and whether AChR cluster growth depends on the presence of agrin. We show that primary myotubes form AChR clusters in response to exogenous agrin in their central region only, a pattern that can spatially restrict NMJ formation. However, the nerve also makes synapses in regions in which aneural AChR clusters do not form, and agrin promotes synaptic cluster growth from the first stages of neuromuscular contact formation. These data indicate that aneural AChR clusters per se are not required for NMJ formation. A model is proposed that explains either the neurocentric or the myocentric mode of NMJ formation depending on a balance between the levels of MuSK expression and the availability of nerve-released agrin.

Induction of multiple signaling loops by MuSK during neuromuscular synapse formation

At the neuromuscular junction, two motor neuron-derived signals have been implicated in the regulation of synaptogenesis. Neuregulin-1 is thought to induce transcription of acetylcholine receptor (AChR) genes in subsynaptic muscle nuclei by activating ErbB receptors. Neural agrin aggregates AChRs by activating the receptor tyrosine kinase MuSK. Here, we show that these two signals act sequentially. Agrin, by activating MuSK, induces the synthesis and aggregation of both MuSK and ErbB receptors. ErbB acts downstream of MuSK in synapse formation. In this way, MuSK activation leads to the establishment of a neuregulin-1-dependent signaling complex that maintains MuSK, ErbB, and AChR expression at the synapse of electrically active muscle fibers.

Muscle-wide secretion of a miniaturized form of neural agrin rescues focal neuromuscular innervation in agrin mutant mice.

Agrin and its receptor MuSK are required for the formation of the postsynaptic apparatus at the neuromuscular junction (NMJ). In the current model the local deposition of agrin by the nerve and the resulting local activation of MuSK are responsible for creating and maintaining the postsynaptic apparatus including clusters of acetylcholine receptors (AChRs). Concomitantly, the release of acetylcholine (ACh) and the resulting depolarization disperses those postsynaptic structures that are not apposed by the nerve and thus not stabilized by agrin-MuSK signaling. Here we show that a miniaturized form of agrin, consisting of the laminin-binding and MuSK-activating domains, is sufficient to fully restore NMJs in agrin mutant mice when expressed by developing muscle. Although miniagrin is expressed uniformly throughout muscle fibers and induces ectopic AChR clusters, the size and the number of those AChR clusters contacted by the motor nerve increase during development. We provide experimental evidence that this is due to ACh, because the AChR agonist carbachol stabilizes AChR clusters in organotypic cultures of embryonic diaphragms. In summary, our results show that agrin function in NMJ development requires only two small domains, and that this function does not depend on the local deposition of agrin at synapses. Finally, they suggest a novel local function of ACh in stabilizing postsynaptic structures.

A minigene of neural agrin encoding the laminin-binding and acetylcholine receptor-aggregating domains is sufficient to induce postsynaptic differentiation in muscle fibres

The extracellular matrix molecule agrin is both necessary and sufficient for inducing the formation of postsynaptic specializations at the neuromuscular junction (NMJ). At the mature NMJ, agrin is stably incorporated in synaptic basal lamina. The postsynapse‐inducing activity of chick agrin, as assayed by its capability of causing aggregation of acetylcholine receptors (AChRs) on cultured muscle cells, maps to a 21 kDa, C‐terminal domain. Binding of chick agrin to muscle basal lamina is mediated by the laminins and maps to a 25 kDa, N‐terminal fragment of agrin. Here we show that an expression construct encoding a ‘mini’‐agrin, in which the laminin‐binding fragment was fused to the AChR‐clustering domain, is sufficient to induce postsynaptic differentiation in vivo when injected into non‐synaptic sites of rat soleus muscle. As shown for ectopic postsynaptic differentiation induced by full‐length neural agrin, myonuclei underneath the ectopic sites expressed the gene for the AChR ε‐subunit. Altogether, our data show that a ‘mini’‐agrin construct encoding only a small fraction of the entire agrin protein is sufficient to induce postsynapse‐like structures that are reminiscent of those induced by full‐length neural agrin or innervation by motor neurons.

Neuregulin signaling is dispensable for NMDA- and GABA(A)-receptor expression in the cerebellum in vivo.

Neuregulin-1s (NRG-1s) are a family of growth and differentiation factors with multiple roles in the development and function in different organs including the nervous system. Among the proposed functions of NRG-1s in the nervous system is the regulation of genes encoding certain neurotransmitter receptors during synapse formation as well as of other aspects of synaptic function. Here, we have examined, in granule cells of the cerebellum in vivo, the role of NRGs in the induction of NMDA receptor (NMDA-R) and GABAA receptor (GABAA-R), which are thought to be induced by NRG-1 secreted by the synaptic inputs. To this end, we used the Cre/loxP system to genetically ablate the NRG receptors ErbB2 and ErbB4 selectively in these cells, thus eliminating all NRG-mediated signaling to them. Unlike previous reports using cultured granule cells to address the same question, we found that the developmental expression patterns of the mRNAs encoding the NR2C subunit of the NMDA-R and the β2-subunit of the GABAA-R is normal in mice lacking the NRG receptors ErbB2 and ErbB4. Likewise, no alterations in cerebellar morphology nor in certain aspects of cerebellar wiring were resolved in these mutants. We conclude that NRG/ErbB signaling to the granule cells is dispensable for the normal development of their synaptic inputs.

Mice Lacking Protease Nexin-1 Show Delayed Structural and Functional Recovery after Sciatic Nerve Crush

Multiple molecular mechanisms influence nerve regeneration. Because serine proteases were shown to affect peripheral nerve regeneration, we performed nerve crush experiments to study synapse reinnervation in adult mice lacking the serpin protease nexin-1 (PN-1). PN-1 is a potent endogenous inhibitor of thrombin, trypsin, tissue plasminogen activators (tPAs), and urokinase plasminogen activators. Compared with the wild type, a significant delay in synapse reinnervation was detected in PN-1 knock-out (KO) animals, which was associated with both reduced proliferation and increased apoptosis of Schwann cells. Various factors known to affect Schwann cells were also altered. Fibrin deposits, tPA activity, mature BDNF, and the low-affinity p75 neurotrophin receptor were increased in injured sciatic nerves of mutant mice. To test whether the absence of PN-1 in Schwann cells or in the axon caused delay in reinnervation, PN-1 was overexpressed exclusively in the nerves of PN-1 KO mice. Neuronal PN-1 expression did not rescue the delayed reinnervation. The results suggest that Schwann cell-derived PN-1 is crucial for proper reinnervation through its contribution to the autocrine control of proliferation and survival. Thus, the precise balance between distinct proteases and serpins such as PN-1 can modulate the overall impact on the kinetics of recovery.

CLASP2-dependent microtubule capture at the neuromuscular junction membrane requires LL5β and actin for focal delivery of acetylcholine receptor vesicles

A novel mechanism is described for the agrin-mediated focal delivery of acetylcholine receptors (AChRs) to the postsynaptic membrane of the neuromuscular junction. Microtubule capture mediated by CLASP2 and its interaction partner, LL5β, and an intact subsynaptic actin cytoskeleton are both required for focal AChR transport to the synaptic membrane.