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

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. Preceding this study, other studies have described the relation between neurotransmission and PKC activity, which, at the same time, are thought to be modulated by pre- and postsynaptic activity in the neuromuscular junction of skeletal muscles. From that premise, we hypothesize that presynaptic and postsynaptic activity regulate the phosphorylation of Munc18-1 and SNAP-25, which play a key role in synaptic vesicle exocytosis. We also hypothesize that this modulation takes place through PKC activity, giving special attention to the exclusively presynaptic isoforms nPKCε and cPKCβI. Moreover, the last one has been related with BDNF/TrkB signaling pathway and, therefore, these could also be involved in Munc18-1 and SNAP-25 regulation. In order to determine this hypothesis, our first objective is to determine the presence of Munc18-1 and SNAP-25 in the peripheral nervous system and their location in the presynaptic terminal to reaffirm their function in the neurotransmission. Then, we will determine if Munc18-1 and SNAP-25 levels are affected after electrical stimulation resulting, or not, in muscle contraction. This will allow us to determine whether Munc18-1 and SNAP-25 are modulated by synaptic activity. Furthermore, because Munc18-1 and SNAP-25 have to be phosphorylated to be active, we will determine whether the presynaptic and/or postsynaptic activity regulates their phosphorylation. Next, we will determine whether Munc18-1 and SNAP-25 phosphorylation depends on Ca2+ and if they are enhanced when PKC family is activated by phorbol esters in the NMJ. Additionally, we will identify whether Munc18-1 and SNAP-25 phosphorylation is related with nPKCε and or cPKCβI, which have been demonstrated to be essential for neurotransmission at the NMJ. Finally, we will determine whether BDNF/TrkB signaling pathway, which is enhanced by synaptic activity and directly related with the cPKCβI isoform in the neuromuscular system, regulates the phosphorylation levels of Munc18-1 and SNAP-25 proteins and whether they are related or not with synaptic activity induced in the skeletal muscle. The main objective of this thesis is to determine how the phosphorylation of the exocytotic proteins Munc18-1 and SNAP-25 is modulated by synaptic activity and muscle contraction through the cPKCβI and nPKCε isoforms and TrkB signaling at the neuromuscular junction. The specific objectives are: 1. To determine the expression, location and regulation by the PKC-activators calcium and PMA of Munc18-1 and SNAP-25 and their phosphorylated forms in the skeletal muscle. 2. To determine whether the synaptic activity modulates Munc18-1, SNAP-25 and their phosphorylation levels in the skeletal muscle through nPKCε and cPKCβI isoforms and BDNF/TrkB signaling pathway. 3. To determine whether the synaptic activity resulting in muscle contraction has repercussion on Munc18-1, SNAP-25 and their phosphorylation levels in the skeletal muscle through nPKCε and cPKCβI isoforms and BDNF/TrkB signaling pathway. 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. Related to SNAP-25, in conclusion, the PKC phosphorylation of the force generator SNAP-25 protein seems a relatively simple process constitutively performed by the nPKCε isoform in basal and different synaptic activity conditions. This PKC isoform also guarantees the phosphorylation of the regulatory Munc18-1 protein. However in this case, a very complex regulation is mediated by at least TrkB and cPKCβI activity, which operate specifically in different conditions of imposed synaptic activity. In regard to SNAP-25, its phosphorylation is not regulated by TrkB nor cPKCβI in any activity conditions.