Coordinated effects of synaptic activity and muscle contraction on cpkc regulation by pdk1 and bdnf/trkb signalling. An approach towards the amyotrophic lateral sclerosis pathophysiology

Resum

The neuromuscular system is a complex and interconnected network in which innervating motoneurons and contacting Schwann cells “tell” skeletal muscle cells to grow, to differentiate and how they should function. Conversely, skeletal muscle provides signals, including neurotrophins, that regulates the survival and function of motoneurons during development, maintenance or injury. Neurotrophins as BDNF, regulate synapse maintenance and function in the neuromuscular system. However, the final BDNF signalling output depends largely on TrkB isoforms (TrkB.FL and TrkB.T1) and P75NTR. BDNF binding to TrkB leads to the activation of PKCs, which are previously maturated by a series of phosphorylation steps, the first of which is mediated by PDK1. How pre- and postsynaptic activities modulates maturation by PDK1 and activation through BDNF/TrkB pathway of presynaptic conventional PKC (cPKCα and/or βI) to modulate synaptic strength at the NMJ, remains unknown. Moreover, BDNF signalling is regulated by activity and it is related with some neuromuscular disorders such as amyotrophic lateral sclerosis (ALS). In ALS, neuromuscular activity is decreased and the link between pre- and postsynaptic components is impaired. Physical exercise by increasing activity has been proposed as a therapy to ameliorate the disease but with contradictory results. In this thesis, we hypothesize that synaptic activity and muscle contraction are closely coordinate to regulate: (1) BDNF/TrkB signalling pathway modulating presynaptic cPKC isoforms (α, βI) to balance synaptic function and (2) PDK1 activation to maturate cPKC in skeletal muscle. In concordance with that, a decreased neuromuscular activity in ALS model mice, changes BDNF levels and induces modifications in its receptors. Moreover, an increased neuromuscular activity induced by exercise prevents the ALS-induced modifications in BDNF signalling. To address that, we have organised this thesis in two chapters. In Chapter I, we study the involvement of synaptic activity and muscle contraction in (1) BDNF/TrkB signalling pathway modulating presynaptic cPKC isoforms (α, βI) to control neurotransmission and (2) PDK1 activation to maturate cPKC in skeletal muscle. To assess that, we stimulated the phrenic nerve of rat diaphragms with or without contraction to differentiate the effects of presynaptic activity from that of muscle contraction. Then, we performed ELISA, Western blotting, qRT-PCR, immunofluorescence and electrophysiological techniques. Results showed that synaptic activity translocated presynaptic pPDK1 to the membrane fraction and increased the phosphorylation of cPKCβI in the same compartment. Once catalytically competent, pcPKCβI and pcPKCα are activated through synaptic activity induced-BDNF/TrkB pathway to enhance ACh release. Consequently, muscle contraction further enhanced the synaptic activity-induced increase of BDNF and decreased TrkB.T1 protein levels, ultimately increasing the ratio TrkB.FL/T1. This led to an increased pool of total PKC ready to be phosphorylated by PDK1, and then, activated by BDNF/TrkB to be involved in neurotransmission. In concordance with that, In Chapter II we study whether BDNF and its receptors (TrkB and p75NTR) are affected in ALS disease and whether the modulation of neuromuscular activity by exercise in a pathological context like ALS, could affect the levels of BDNF, TrkB and p75NTR. To determine that, we performed running vs swimming-based training protocols to analyse the BDNF/TrkB signalling pathway in the plantaris muscle of SOD1-G93A mice by Western Blot. In ALS context, where muscle contraction is decreased, BDNF signalling is impaired manly due to the dysregulation of its receptors. mBDNF levels increase while p75NTR, TrkB.FL, pTrkB.FL levels decrease and TrkB.T1 levels increase. However, when muscle contraction is increased through different physical exercise imposed in ALS mice (running or swimming-based training) the impairment of BDNF signalling is prevented in a different way depending on the nature and intensity of the exercise. Thus, in this thesis we demonstrate that both synaptic activity and muscle contraction are closely coordinated and regulated in a complex and balanced way to preserve NMJ function. Further basic research is needed to understand the way that NMJ works and why it matters. It will provide the foundation of knowledge for the applied clinical science that follows. Thus, it will allow us a better understanding of neuromuscular disorders in which there is a progressive loss of the connection between nerve and muscle with an impaired NMJ function.