BDNF/TrkB signalling, in cooperation with muscarinic signalling, retrogradely regulates PKA pathway to phosphorylate SNAP-25 and Synapsin-1 at the neuromuscular junction

• Published in: Cell communication and signaling 2024-07, Vol.22 (1), p.371-20, Article 371
• Main Authors: Polishchuk, Aleksandra, Cilleros-Mañé, Víctor, Balanyà-Segura, Marta, Just-Borràs, Laia, Forniés-Mariné, Anton, Silvera-Simón, Carolina, Tomàs, Marta, Jami El Hirchi, Meryem, Hurtado, Erica, Tomàs, Josep, Lanuza, Maria A
• Format: Article
• Language: English
• Subjects: Acetylcholine; Acetylcholine receptors (muscarinic); Acetylcholine release; Analysis; Animals; Antibodies; Autocrine signalling; BDNF; Brain-derived neurotrophic factor; Brain-Derived Neurotrophic Factor - metabolism; Care and treatment; Catalytic subunits; Communication; Cooperation; Cyclic AMP-Dependent Protein Kinases - metabolism; Diagnosis; Diaphragm; Drug therapy; Exocytosis; Health aspects; Immunohistochemistry; Kinases; Laboratory animals; Male; Marine toxins; Molecular mechanics; Molecular modelling; Motor endplate; Muscarinic receptors; Muscle contraction; Neuromodulation; Neuromuscular diseases; Neuromuscular Junction - metabolism; Neuromuscular junctions; Neurotransmission; Phosphorylation; Phrenic nerve; Physiological aspects; Pirenzepine; Protein kinase A; Proteins; Rats; Rats, Wistar; Receptor, trkB - metabolism; Receptors, Muscarinic - metabolism; Regulatory subunits; Risk factors; Serine kinases; Signal Transduction; Synapses; Synapsin; Synapsins - metabolism; Synaptic plasticity; Synaptosomal-Associated Protein 25 - metabolism; Toxins; TrkB; TrkB receptors; Viral antibodies; Western blotting
• ISSN: 1478-811X; 1478-811X
• DOI: 10.1186/s12964-024-01735-2

Protein kinase A (PKA) enhances neurotransmission at the neuromuscular junction (NMJ), which is retrogradely regulated by nerve-induced muscle contraction to promote Acetylcholine (ACh) release through the phosphorylation of molecules involved in synaptic vesicle exocytosis (SNAP-25 and Synapsin-1). However, the molecular mechanism of the retrograde regulation of PKA subunits and its targets by BDNF/TrkB pathway and muscarinic signalling has not been demonstrated until now. At the NMJ, retrograde control is mainly associated with BDNF/TrkB signalling as muscle contraction enhances BDNF levels and controls specific kinases involved in the neurotransmission. Neurotransmission at the NMJ is also highly modulated by muscarinic receptors M and M (mAChRs), which are related to PKA and TrkB signallings. Here, we investigated the hypothesis that TrkB, in cooperation with mAChRs, regulates the activity-dependent dynamics of PKA subunits to phosphorylate SNAP-25 and Synapsin-1. To explore this, we stimulated the rat phrenic nerve at 1Hz (30 minutes), with or without subsequent contraction (abolished by µ-conotoxin GIIIB). Pharmacological treatments were conducted with the anti-TrkB antibody clone 47/TrkB for TrkB inhibition and exogenous h-BDNF; muscarinic inhibition with Pirenzepine-dihydrochloride and Methoctramine-tetrahydrochloride for M and M mAChRs, respectively. Diaphragm protein levels and phosphorylation' changes were detected by Western blotting. Location of the target proteins was demonstrated using immunohistochemistry. While TrkB does not directly impact the levels of PKA catalytic subunits Cα and Cβ, it regulates PKA regulatory subunits RIα and RIIβ, facilitating the phosphorylation of critical exocytotic targets such as SNAP-25 and Synapsin-1. Furthermore, the muscarinic receptors pathway maintains a delicate balance in this regulatory process. These findings explain the dynamic interplay of PKA subunits influenced by BDNF/TrkB signalling, M and M mAChRs pathways, that are differently regulated by pre- and postsynaptic activity, demonstrating the specific roles of the BDNF/TrkB and muscarinic receptors pathway in retrograde regulation. This complex molecular interplay has the relevance of interrelating two fundamental pathways in PKA-synaptic modulation: one retrograde (neurotrophic) and the other autocrine (muscarinic). This deepens the fundamental understanding of neuromuscular physiology of neurotransmission that gives plasticity to synapses and ho