Effects of neuromuscular activity coupled to BDNF/TrkB signaling on the phosphorylation of the exocytotic proteins Munc18-1 and SNAP-25 through nPKCε and cPKCβI

Identificador:  TDX:2706

Handle: https://hdl.handle.net/20.500.11797/TDX2706

Autores:  Simó Ollé, Anna

Resumen:
At the neuromuscular junction (NMJ) synapse, several signaling pathways coordinate pre-, post-synaptic responses and associated glial cells. The relation between these signaling pathways modulates the pool of synaptic vesicles leading to neurotransmission. Moreover, PKC phosphorylates several molecules of synaptic vesicle exocytotic apparatus responsible to the regulation of neurotransmitter release. Munc18-1 and SNAP-25 are two PKC substrates that play a key role in the exocytotic machinery. In addition, PKC is modulated by presynaptic and postsynaptic activity in skeletal muscles. Nevertheless, it is still unknown which PKC regulates these key molecules in the NMJ. cPKCβI and nPKCƐ are exclusively located at the nerve terminal of the NMJ and are regulated by synaptic activity. In addition, muscle contraction through BDNF/TrkB has an important impact on these PKC isoforms. Therefore, this thesis is aimed to determine the expression, location and regulation by the PKC-activators calcium and phorbol esters (PMA) of Munc18-1 and SNAP-25 and their phosphorylated forms in the skeletal muscle. Also, to study whether Munc18-1 and SNAP-25 phosphorylation are affected by (1) synaptic activity and muscle contraction per se; and (2) nPKCƐ, cPKCβI and BDNF/TrkB signaling in a neuromuscular activity-dependent manner. Main results, obtained by Western blot analysis and confocal microscopy, show that Munc18-1 and SNAP-25 are expressed and phosphorylated in basal conditions in the skeletal muscle, predominantly in the membrane fraction, with Munc18-1 being located at the nerve terminal. Munc18-1 and SNAP-25 phosphorylation are modulated by calcium, PMA, synaptic activity and enhanced by nPKCƐ. Otherwise, cPKCβI and BDNF/TrkB signaling pathway regulates Munc18-1 but not SNAP-25 phosphorylation. Finally, muscle contraction downregulates these proteins to reach a basal state. In conclusion, these results provide a mechanistic insight into how Munc18-1 and SNAP-25 phosphorylation is regulated to achieve the extraordinary precision and plasticity of neurotransmission.