The Neuromuscular System: Postsynaptic Regulation

Abstract

Synapses are specialized structures for neural functions at both the peripheral and central nervous system. The efficiency of synaptic transmission between the presynaptic and postsynaptic cells depends on the maintenance of an appropriate receptor density at postsynaptic sites. Despite extensive studies on synaptic plasticity, the mechanisms that regulate the density of postsynaptic neurotransmitter receptors is not fully understood. In this thesis, I used the peripheral neuromuscular junction (NMJ), a chemical synapse made between a spinal motor neuron and skeletal muscle fibers, as a model to investigate the interplay between postsynaptic proteins and acetylcholine receptors (AChRs).

A high density of AChRs at postsynaptic membranes is the hallmark of NMJs. Two major groups of proteins are critical for the tethering and stabilization of AChRs: the agrin-Lrp4/MuSK/rapsyn signaling pathway and the auxiliary proteins [dystrophin-glycoprotein complex (DGC), neuregulin molecules, and the non-kinase muscle-specific anchoring protein (αkap)]. Recent work from our lab has shown that αkap forms a complex with AChRs and promotes their stability by an ubiquitin-dependent mechanism. However, it remains unclear the interplay between the postsynaptic protein network and the stability of AChRs. To understand the connection between the stability/function of AChRs and postsynaptic proteins, I have used a multi-disciplinary approach, including biochemical and molecular tools, mouse models and cultured cell lines.

First, in collaboration with Dr. Martinez, we explored the mechanism by which rapsyn, a core protein for AChR clustering, is targeting the synaptic membrane for its function. By electroporating rapsyn domain deletion mutants-GFP into sternomastoid muscles, we found that the targeting of rapsyn is independent of synaptic activity neither the N-terminal myristoylation nor the RING-H2 domain of rapsyn. However, when the AChR-binding coiled-coil domain is deleted, rapsyn fails to associate with AChRs at NMJs of living mice, providing evidence for an active role of AChRs in the targeting of rapsyn to NMJs.

I also investigated α-dystrobrevin, a cytoplasmic protein of the DGC, on the expression of αkap and its association with AChRs. α-Dystrobrevin is required for the maintenance of a high postsynaptic receptor density and maturation of NMJs. My results established a strong link between the expression levels of αkap and α-dystrobrevin on the expression of AChRs. Particularly, I showed that α-dystrobrevin forms a complex with αkap and promotes its expression in a dose-dependent manner, while the expression of α-dystrobrevin is independent of αkap. Importantly, α-dystrobrevin and αkap promote AChR accumulation and enhance the size of agrin-induced AChR clusters, which suggests the protective role of αkap on AChR is mediated by α-dystrobrevin. To further explore how αkap/α-dystrobrevin stabilizes AChRs, I investigated the deubiquitinating enzyme Usp9x, an α-dystrobrevin binding protein. My results showed that Usp9x promotes the αkap/α-dystrobrevin stabilizing effect on AChRs and its expression is highly correlated with the maturation of synapses.

Finally, Dr. Martinez and I generated α-syntrophin/α-dystrobrevin null mouse to investigate signaling and structural functions of these proteins in the maturation and stability of NMJs. Analyses of these mice revealed that these two proteins do not complement each other and suggested that they may function in the same signaling pathway.

Collectively, my results showed a mechanistic link between the intracellular proteins, rapsyn, α-dystrobrevin, α-syntrophin, αkap, and Usp9x and the stability of the postsynaptic AChR and ultimately the structural integrity of the NMJ. Such findings are potentially important for adding new insights into discovering new therapeutic targets of NMJ-related disorders.