RNA interference: a new tool to study gene functions in adult mammalian muscle "in vivo"

Abstract

RNA interference (RNAi) is a powerful method for sequence-specific posttranscriptional gene silencing (PTGS), which allows rapid survey of gene functions using double-stranded RNA (dsRNA). At the time when we started this work, RNAi was a recently developed tool that had been successfully applied to many organisms, in particular C. elegans and Drosophila, but not to any mammalian system. It was generally doubted that RNAi would also work in mammals in vivo, because the introduction of dsRNA can induce general shutdown of translation and apoptosis in several mammalian cell types. One excellent model system for investigating this open question is the nervemuscle synapse known as the neuromuscular junction (NMJ). Characteristic for the NMJ is the precise apposition of the neurotransmitter release machinery on the nerve terminal side and the neurotransmitter receptors on the muscle fiber membrane. At least two mechanisms underlie the formation and maintenance of a postsynaptic apparatus on the muscle fiber membrane. Both mechanisms are triggered by the heparan sulfate proteoglycan agrin, which is released by the motor neuron. First, neural agrin activates all the cellular mechanisms necessary to assemble a fully functional postsynaptic structure including aggregates of acetylcholine receptors (AChRs). Besides this redistribution of preexisting molecules, agrin signaling restricts the transcription of postsynaptic proteins to myonuclei located in the NMJ. Still little is known about the agrin signaling cascade. Therefore, once RNAi could be developed for mammals system, it will in turn provide a unique tool to address the role of newly identified genes in the postsynaptic differentiation, since there are no tools available for the fast and reliable perturbation of gene function in vivo. In the first part of this work, we investigated the potential of RNAi in perturbing the formation and stability of postsynaptic structures in adult muscle in vivo (chapter 2 and 3). First, we used the experimental paradigm where neural agrin expressed in nonjunctional regions of rat soleus muscle induces formation of ectopic AChR aggregates. Knockout experiments have shown that this agrin activity requires the receptor tyrosine kinase MuSK and the AChR-associated scaffolding molecule rapsyn, but not the cytoskeletal proteins sarcoglycan α (SGCA) and utrophin. In our experiments, we show that co-injection of dsRNAs derived from MuSK or rapsyn perturbed agrin-induced formation of ectopic AChR aggregates, while dsRNAs derived from SGCA or utrophin had no significant effect. In a further step, we used RNAi to study the role of MuSK at adult NMJs. Here, the electroporation of plasmids encoding short hairpin-based 21-bp small interfering RNAs (siRNAs) or long hairpin dsRNAs, which allow global and sustained perturbation of MuSK expression, leads to the disassembly of NMJs in adult mice. These results are consistent with the finding that auto-antibodies to MuSK, which also lower the amount of MuSK protein, cause severe forms of myasthenia gravis. In summary, these results demonstrate for the first time the effectiveness of long dsRNA as well as siRNA in silencing endogenous genes in adult mammalian muscle in vivo and they provide strong evidence that continuous expression MuSK is required to maintain the NMJ. The second part of this work aimed to establishing RNAi in adult muscle to study the role of newly identified genes in the development of the NMJ and in the growth of muscle fibers (chapter 4 and appendix). First, we used RNAi to perturb neural agrininduced formation of ectopic AChR aggregates on mouse soleus muscle. We show that electroporation of plasmids encoding short hairpin- derived siRNA for MuSK leads to the perturbation of ectopic AChR aggregation, regardless whether agrin expression vector or recombinant protein was applied to innervated or denervated muscles. These results clearly show the reliability of RNAi in adult muscle in vivo and therefore set the stage for experiments aimed to study the function of genes, whose expression is altered during the formation postsynaptic structures. A protocol was established to identify functional siRNA target sites in several genes. Plasmids were designed that encoded short hairpin RNAs (shRNAs) derived from different putative effectors of the mammalian target of rapamycin (mTOR) signaling pathway. For some candidate effectors, electroporation of the corresponding plasmids into mouse soleus muscle leads to altered muscle fiber size. These preliminary results are consistent with several reported findings, which indicate that the mTOR signaling pathway is a central controller of muscle fiber atrophy and hypertrophy. The efficiently induced RNAi in those experiments demonstrates that our protocol is useful for identifying siRNA targets. In summary, these results demonstrate that we have successfully established RNAi as a fast and reliable gene knockdown method in muscle fibers of mammals. This method will be important for future investigation of gene functions in adult mammalian muscle in vivo.