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Molecular mechanisms involved in synapse formation and neuromuscular disease

The overall research topic concerns the molecular mechanisms important for the function and dysfunction of synapses at the neuromuscular junction (NMJ) and between neurons in the brain (Figure 1). Pathological changes at the NMJ, as for example seen in myasthenia gravis (Figure 2or muscle dystrophies (Figure 3), impair muscle function and can be life-threatening. Our group investigates different signaling pathways important for the establishment and function of synapses. Using this knowledge, we also try to find new ways to treat pathological alterations at the NMJ and in skeletal muscle. All these projects synergize with each other as we are using the same systems to answer related questions.

The role of mTORC1 and mTORC2 in brain and skeletal muscle
Both, neurons and skeletal muscle fibers are postmitotic and thus their growth largely depends on changes in cell size and not cell number. The mammalian target of rapamycin (mTOR), which was discovered in yeast by Michael Hall and collaborators, assembles into two multiprotein complexes called mTOR complex 1 (mTORC1) and mTORC2. The two complexes are characterized by the presence of particular proteins that are necessary for their function, such as raptor (mTORC1) and rictor (mTORC2). While mTORC1 is inhibited by the immunosuppressant rapamycin, mTORC2 is not inhibited by this drug. We are investigating the role of mTORC1 and mTORC2 in brain and skeletal muscle in mice using the Cre/loxP technology. In both tissues, deletion of mTORC1 causes the organ to be smaller. In the brain, mTORC1 is essential for brain development while mTORC2 mutants survive. In the brain, mTORC2 deficiency causes a strong decrease in its size and the mice display behavioral abnormalities. Neurons show deficits in dendritic arborization and synaptic circuitry. We are currently investigating the detailed molecular mechanisms underlying these phenotypes as well as the function of mTORC1 and mTORC2 at adult synapses.

Like in the brain, mTORC1 deletion in skeletal muscle results in a more severe phenotype than mTORC2 deletion. Here, mTORC2 deletion does not cause any overt phenotype whereas mTORC1 mutation affects the metabolism and function of skeletal muscle. Importantly, mTORC1 deficiency causes a severe myopathy and leads to the death of the mice at the age of 4 to 6 months. Moreover, the mice display a general wasting syndrome that is not restricted to skeletal muscle. Current projects study the mechanisms that trigger this wasting and investigate the contribution of mitochondrial biogenesis to the overall phenotype. In addition, we examine the effect of activating mTORC1 in skeletal muscle by the deletion of its inhibitor TSC1. 

Copine 6, a novel calcium sensor involved in synapse structure
Changes in synaptic activity alter synaptic transmission and ultimately change neuronal network dynamics. Structural changes induced by electrical activity are often mediated by calcium-dependent processes. Initiated by gene expression studies at the NMJ, we focus on the function of Copine 6, a member of a small family of calcium-binding proteins. We have shown that Copine 6 affects spine formation and maintenance in hippocampal neurons. Copine 6 shuttles from the cytosol to postsynaptic sites upon NMDA receptor-dependent calcium influx. It binds to the Rho GTPase Rac1 and mediates its translocation to membranes upon calcium influx. These results strongly suggest that Copine 6 serves as a calcium sensor that links neuronal activity to the subsequent changes in synaptic structure. In the current projects, we investigate the function of Copine 6 and additional family members in vivo and we try to identify additional binding partners.

NMJ and disease
There are sporadic and genetic neuromuscular diseases (NMDs). Although they are often severe, they affect only a small proportion of the human population and most of the diseases are still not treatable. One of the acquired NMDs is myasthenia gravis that is caused by auto-immune antibodies directed to components of the NMJ. While most of the antibodies are directed against the acetylcholine receptor, in about 10% of the cases antibodies are directed against the receptor tyrosine kinase MuSK. Interestingly, the clinical symptoms are clearly distinct between the two subgroups. We have recently shown that the phenotypic difference between the two subtypes of myasthenia gravis can also be reiterated in a murine model of experimental autoimmune myasthenia gravis (EAMG). The symptoms in MuSK-EAMG mice are a severe kyphosis, weight loss and signs of neuromuscular hyperactivity, which are all distinct from the symptoms in EAMG induced by acetylcholine receptor antibodies. At the NMJs, MuSK-EAMG causes fragmentation and often loss of innervation (Figure 2). Interestingly, like in human patients, the pathological changes observed at NMJs differ between muscles. Current studies aim at elucidating the molecular mechanisms that are responsible for this difference between muscles.

Another set of NMDs are muscular dystrophies, where the skeletal muscle fibers and not the NMJs are affected. Although the mutations causing muscular dystrophies are distinct, the severe loss of muscle mass due to the degeneration of muscle fibers is common to all diseases. As a consequence, muscle becomes replaced by fibrotic tissue (Figure 3A). In one project we develop new methods to treat some of the muscular dystrophies in experimental mouse models. For example, we have recently shown that transgenic expression of a miniaturized form of the extracellular matrix molecule agrin (mini-agrin) can substantially compensate for the loss of laminin-211. This compensation is even more complete when cell death of muscle fibers is prevented by additionally expressing the anti-apoptotic protein Bcl2 (Figure 3B) or treating mice with the anti-apoptotic agent omigapil. Such combination therapy restores the structure of the muscle to control levels (Figure 3C). In current projects we test additional pharmacological agents for their potential to ameliorate the disease.

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