The TrkB signalling at the neuromuscular junction of amyotrophic lateral sclerosis mice. The protective effect of exercise.

Abstract

The bidirectional communication between the presynaptic and postsynaptic elements at the neuromuscular junction (NMJ) guarantees its health and optimal function. Consequently, a well-preserved NMJ positively impacts on its cellular components condition, including the motor neuron, the myocyte and the terminal Schwann cell. In accordance, changes in the neuromuscular activity induce plasticity mechanisms that modify NMJ basal function, resulting in either neuromuscular dysfunction or reinforcement. The TrkB signalling pathway is strongly related with NMJ plasticity. In particular, the neurotrophins BDNF and NT-4, that are released in response to neuromuscular activity, activate the TrkB receptor. At its turn, TrkB regulates kinases like PKC and PKA, that phosphorylate molecules implicated in the exocytotic machinery, such as Munc18-1 and SNAP-25. In consequence, changes in the presynaptic activity and the resulting muscle contraction induce adjustments in the TrkB signalling pathway in a feedback loop that modulates neurotransmission at the NMJ to promote neuromuscular health and the protection of the cell partners. Therefore, understanding how activity changes influence the TrkB signalling pathway at the NMJ could contribute to the development of therapeutic strategies for neurological diseases concerning NMJ formation, maintenance and function. For that reason, this thesis aims to unveil the relation between the TrkB signalling pathway plasticity and the protection of the neuromuscular and locomotor system. We hypothesize that the TrkB signalling pathway works differently depending on the properties of each muscle, which condition their selective vulnerability to neurodegenerative diseases such as Amyotrophic lateral sclerosis (ALS). Thus, in a vulnerable muscle, TrkB signalling alterations should be found in correlation with decreased neuroprotective capacity at the NMJ and with the loss of the tightly regulated nerve-muscle bidirectional communication, resulting in motor impairment, muscle atrophy and paralysis. Furthermore, we expect that increased NMJ activity mediated by exercise would recover the ALS-induced TrkB signalling alterations in relation with an improvement in muscular innervation and function. Finally, analysing the TrkB signalling pathway in trained healthy mice would allow to determine how TrkB mediates for the beneficial effects of physical exercise. To confirm these hypotheses, we have determined I) the differences in the TrkB signalling between fatigue-resistant and fatigable limb muscles and extra-ocular muscles in relation with their vulnerability to denervation; II) the differences in the TrkB signalling derived from the two long-term training protocols in a healthy muscle vulnerable to ALS in relation with fatigue resistance acquisition; III) the alterations in the TrkB signalling in an ALS vulnerable muscle in relation with MN loss; and IV) the prevention effect of two long-term training protocols in an ALS vulnerable muscle over MN loss and TrkB signalling. To achieve these objectives, the present thesis has analysed by Western blotting the entire downstream TrkB signalling, thus, concerning I) the BDNF and NT-4 neurotrophins, II) the neurotrophins receptors p75NTR and TrkB isoforms, III) the downstream kinases PDK1, PKC , PKC I and PKC isoforms and PKA catalytic and regulatory subunits and IV) Munc18-1 and SNAP-25, two PKC and PKA targets implicated in synaptic exocytosis and neurotransmission. Furthermore, we have analysed the MNs population distribution under different activity conditions. Results show that healthy exercised myocytes synthesize a pool of proBDNF that can be cleaved into mBDNF on demand, following an intensity dependent fast-to-slow molecular transition. However, proBDNF synthesis due to exercise does not occur in ALS mice, pointing to a lack of protein synthesis in these mice. Consequently, the TrkB signalling is strongly perturbed in muscles that are especially vulnerable to ALS. In particular, the shorter, negative TrkB.T1 isoform is predominant over the longer, positive, TrkB.FL isoform, even before mice show any symptom of the disease in the Plantaris muscle, resulting in an accumulation of mBDNF that cannot trigger its signal. On the other hand, exercise promotes TrkB.FL synthesis and the downstream signalling in healthy and ALS mice, evidenced by mBDNF consumption. This molecular recovering coincides with reduced muscular atrophy and less MN loss in trained mice. On the other hand, NT-4 is upregulated in ALS mice, pointing that its expression corresponds to the surviving slower fibres of the fast Plantaris muscle. Altogether, elevated levels of muscle derived neurotrophins confirm that decreased neurotrophic support from muscle tissue is not the cause of NMJ degeneration in ALS, but neurotrophin receptors impairment. However, results after exercise point to the opportunity to modulate endogenous neurotrophins beneficial effects as it can reactivate their effect, proportionally to the intensity of the applied exercise. These results also explain the fact that fatigue resistant muscles are also more resistant to ALS disease, as they have a higher neuroprotective capacity due to a more intense TrkB signalling. Furthermore, they justify the beneficial effects of exercise, proportional to the intensity of the protocol, on fast muscles as it promotes pTrkB.FL consumption following a gain of resistance. Downstream the TrkB.FL receptor, the results show a regular pPDK1 consumption in fatigue resistant and trained muscles, confirming its constitutive activity. However, differential PKC activity is detected through muscles under basal conditions. In brief, classic PKC expression is predominant in fast muscles, thus inversely proportional to fatigue resistance while novel PKC expression is predominant in slow muscles, proportional to fatigue resistance, in non-trained different muscles. In accordance, cPKC and cPKC I expression and activity decrease after swimming, in the context of a metabolic fast-to-slow molecular transition. On the other hand, either PDK1 or pPKC I are not working in ALS mice, but its inactivity is restored by exercise, pointing to a reduction in the fast-to-slow pathologic transition and decreased MN loss. Following constant PKC activity through muscles, Munc18-1 and pMunc18-1 maintain their ratio under basal activity conditions, On the other hand, exercise promotes Munc18-1 consumption in both WT and ALS mice. Thus, pMunc18-1 is accumulated in ALS due to decreased postsynaptic activity, but exercise normalizes it proportionally to protocol intensity. Similarly, SNAP-25 expression is proportional to fatigue resistance in the studied muscles under basal conditions while pSNAP-25 (S187) is low in extra-ocular muscles and after swimming in WT mice, pointing to an intense consumption in these muscles. By the contrary, pSNAP-25 (S187) is accumulated in ALS mice, even despite of exercise protocols, in accordance with a reduction in neurotransmission. On the other hand, PKA catalytic and regulatory subunits proportion is similar through healthy limb muscles. By the contrary, ALS affected Plantaris muscles show increased catalytic subunits expression and pSNAP-25 (T138) accumulation even despite of exercise protocols. Thus, exercise does not avoid PKA target over-phosphorylation in ALS mice. Otherwise, pSNAP-25 accumulation seems to be related with the pathology, pointing to a lack of degradation. Altogether, we confirm that under healthy conditions, exercise promotes a fast-to-slow transition that favours a gain of resistance in fatigable muscles through TrkB signalling modulation. On the other hand, the TrkB signalling is strongly altered by ALS and exercise partially restores it by preventing a pathologic fast-to-slow transition.