Muscarinic receptor modulation of protein kinase A, protein kinase C and exocytotic proteins at the neuromuscular junction

La unió neuromuscular (NMJ) utilitza mecanismes de plasticitat per adequar l’alliberament d’acetilcolina (ACh) dins d’un entorn molt dinàmic. Els receptors muscarínics d’acetilcolina (mAChRs) participen com a autoreceptors, ajustant la neurotransmissió. El subtipus M1 activa la proteïna quinasa C (PKC) per potenciar la transmissió, mentre que l’M2 inhibeix la proteïna quinasa A (PKA) per reduir-la. En la passada dècada, grans descobriments ens han aportat un coneixement electrofisiològic extens sobre la senyalització muscarínica. Tanmateix, les dades moleculars segueixen sent escasses i el rol d’alguns segons missatgers resta desconegut. Així doncs, aquesta tesi es realitza amb l’objectiu de caracteritzar com els receptors M1 i M2 modulen les isoformes de PKA i PKC, les seves proteïnes reguladores i les dianes d’exocitosi. Hem analitzat la cascada muscarínica al múscul diafragma de rata usant inhibidors selectius i generals de M1, M2, nPKCε, cPKCβI, PKA i PDK1 i analitzant cada alteració mitjançant Western blot, així com fraccionament subcel·lular i co-immunoprecipitació. També hem fet ús de tècniques immunohistoquímiques i microscòpia confocal per corroborar la localització presinàptica de les molècules d'interès. Els resultats mostren que els nivells del receptor M1 són disminuïts per la via de l’M2. Respecte a la senyalització de la PKA, l’M2 inhibeix la seva activitat regulant la subunitat Cβ a la baixa, la subunitat RIIα/β a l’alta i alliberant RIβ i RIIα al citosol, el que redueix la fosforilació de SNAP-25 (Thr138) i CREB. L’M1 s’interposa en la senyalització M2/PKA reincorporant les subunitats R a la membrana. Respecte la senyalització PKC, ambdós M1 i M2 poden activar la quinasa mestra PDK1, que promou la maduració de les isoformes de PKC βI i ε presinàptiques. L’M1 recluta les dues PKC madures a la membrana i promou la fosforilació de Munc18-1, SNAP-25 i MARCKS. Al contrari, l’M2 regula a la baixa la PKCε de forma dependent de PKA, el que inhibeix la síntesi de Munc18-1 i la seva fosforilació. El treball present contribueix a comprendre l’acció conjunta i interdependent dels receptors M1 i M2 per regular la neurotransmissió.La unión neuromuscular (NMJ) utiliza mecanismos de plasticidad para adecuar la liberación de acetilcolina (ACh) a un entorno muy dinámico. Los receptores muscarínicos de acetilcolina (mAChRs) participan como autorreceptores, ajustando la neurotransmisión. El subtipo M1 activa la proteína quinasa C (PKC) para potenciar la transmisión, mientras que el M2 inhibe la proteína quinasa A (PKA) para reducirla. La interesante investigación de la pasada década nos ha aportado un extenso conocimiento electrofisiológico sobre la señalización muscarínica. Aun así, los datos moleculares siguen siendo escasos y el rol de algunos segundos mensajeros permanece desconocido. Así pues, esta tesis tiene el objetivo de caracterizar cómo los receptores M1 y M2 regulan las isoformas de PKA y PKC, sus proteínas reguladoras y las dianas de exocitosis. Para ello, hemos analizado la cascada muscarínica en el músculo diafragma de rata usando inhibidores selectivos y generales de M1, M2, nPKCε, cPKCβI, PKA y PDK1 y analizando dichas alteraciones mediante Western blot, fraccionamiento subcel·lular y co-inmunoprecipitación. También hemos hecho uso de técnicas inmunohistoquímicas y confocales para corroborar la localización presináptica de nuestras moléculas de interés. Los resultados muestran que los niveles del receptor M1 son disminuidos por la vía del M2. Respecto a la señalización PKA, M2 inhibe su actividad total disminuyendo la subunidad Cβ, aumentando las subunidades RIIα/β y liberando a RIβ y RIIα al citosol, lo que reduce la fosforilación de SNAP-25 (Thr138) y CREB. El receptor M1 se interpone en la señalización M2/PKA reincorporando las subunidades R a la membrana. Respecto la señalización PKC, ambos M1 y M2 pueden activar la quinasa maestra PDK1, que promueve la maduración de las isoformas PKCβI y ε presinápticas. M1 recluta las dos PKC maduras a la membrana y promueve la fosforilación de Munc18-1, SNAP-25 y MARCKS. Al contrario, el M2 inhibe la PKCε de forma dependiente de PKA, lo que también disminuye la síntesis de Munc18-1 y su fosforilación. Este trabajo contribuye a comprender la acción conjunta e interdependiente de los receptores M1 y M2 sobre la neurotransmisión.The neuromuscular junction (NMJ) uses plastic mechanisms to adjust the release of acetylcholine (ACh) to an incredibly dynamic environment. Muscarinic acetylcholine receptors (mAChRs) participate as autoreceptors, tuning neurotransmission. The M1 subtype activates protein kinase C (PKC) to enhance the release, whereas M2 inhibits protein kinase A (PKA) to decrease it. The captivating research in the past decade has provided extensive electrophysiological knowledge about muscarinic signaling. However, the molecular data accompanying this knowledge was limited; and the role of some second messengers remained elusive. Therefore, the present thesis aimed to characterize how M1 and M2 mAChRs regulate the multiple PKA and PKC subunits, their scaffolds and exocytotic targets. We analyzed the muscarinic cascade at the rat diaphragm muscle by testing selective and general inhibitors of M1 and M2 mAChR, nPKCε, cPKCβI, PKA and PDK1 and analyzed each alteration mainly by Western blotting as well as subcellular fractionation and co-immunoprecipitation. We also made use of immunohistochemical and confocal techniques to corroborate the presynaptic location of our molecules of interest. Our results show that M1 receptors are downregulated by the M2 pathway. Regarding PKA signaling, M2 inhibits PKA activity by downregulating Cβ subunit, upregulating RIIα/β and liberating RIβ and RIIα to the cytosol, which reduces the phosphorylation of SNAP-25 on Thr138 and CREB. M1 signaling crosstalks with M2/PKA by recruiting R subunits to the membrane. Regarding PKC signaling, both M1 and M2 mAChR activate the master kinase PDK1, which promotes the priming of the presynaptic PKCβI and PKCε isoforms. M1 recruits both primed PKCs to the membrane and promotes Munc18-1, SNAP-25 and MARCKS phosphorylation. In contrast, M2 downregulates PKCε through a PKA-dependent pathway, which inhibits Munc18-1 synthesis and its PKC-phosphorylation. The results demonstrate that M1 and M2 mAChRs perform a coordinated and interdependent signaling to modulate neurotransmission at the NMJ

Running and Swimming Differently Adapt the BDNF/TrkB Pathway to a Slow Molecular Pattern at the NMJ

International audiencePhysical exercise improves motor control and related cognitive abilities and reinforces neuroprotective mechanisms in the nervous system. As peripheral nerves interact with skeletal muscles at the neuromuscular junction, modifications of this bidirectional communication by physical activity are positive to preserve this synapse as it increases quantal content and resistance to fatigue, acetylcholine receptors expansion, and myocytes’ fast-to-slow functional transition. Here, we provide the intermediate step between physical activity and functional and morphological changes by analyzing the molecular adaptations in the skeletal muscle of the full BDNF/TrkB downstream signaling pathway, directly involved in acetylcholine release and synapse maintenance. After 45 days of training at different intensities, the BDNF/TrkB molecular phenotype of trained muscles from male B6SJLF1/J mice undergo a fast-to-slow transition without affecting motor neuron size. We provide further knowledge to understand how exercise induces muscle molecular adaptations towards a slower phenotype, resistant to prolonged trains of stimulation or activity that can be useful as therapeutic tools

BDNF-TrkB Signaling Coupled to nPKCε and cPKCβI Modulate the Phosphorylation of the Exocytotic Protein Munc18-1 During Synaptic Activity at the Neuromuscular Junction

Munc18-1, a neuron-specific member of the Sec1/Munc18 family, is involved in neurotransmitter release by binding tightly to syntaxin. Munc18-1 is phosphorylated by PKC on Ser-306 and Ser-313 in vitro which reduces the amount of Munc18-1 able to bind syntaxin. We have previously identified that PKC is involved in neurotransmitter release when continuous electrical stimulation imposes a moderate activity on the NMJ and that muscle contraction through TrkB has an important impact on presynaptic PKC isoforms levels, specifically cPKCβI and nPKCε. Therefore, the present study was designed to understand how Munc18-1 phosphorylation is affected by (1) synaptic activity at the neuromuscular junction, (2) nPKCε and cPKCβI isoforms activity, (3) muscle contraction per se, and (4) the BDNF/TrkB signaling in a neuromuscular activity-dependent manner. We performed immunohistochemistry and confocal techniques to evidence the presynaptic location of Munc18-1 in the rat diaphragm muscle. To study synaptic activity, we stimulated the phrenic nerve (1 Hz, 30 min) with or without contraction (abolished by μ-conotoxin GIIIB). Specific inhibitory reagents were used to block nPKCε and cPKCβI activity and to modulate the tropomyosin receptor kinase B (TrkB). Main results obtained from Western blot experiments showed that phosphorylation of Munc18-1 at Ser-313 increases in response to a signaling mechanism initiated by synaptic activity and directly mediated by nPKCε. Otherwise, cPKCβI and TrkB activities work together to prevent this synaptic activity–induced Munc18-1 phosphorylation by a negative regulation of cPKCβI over nPKCε. Therefore, a balance between the activities of these PKC isoforms could be a relevant cue in the regulation of the exocytotic apparatus. The results also demonstrate that muscle contraction prevents the synaptic activity–induced Munc18-1 phosphorylation through a mechanism that opposes the TrkB/cPKCβI/nPKCε signaling

Running and Swimming Differently Adapt the BDNF/TrkB Pathway to a Slow Molecular Pattern at the NMJ

Physical exercise improves motor control and related cognitive abilities and reinforces neuroprotective mechanisms in the nervous system. As peripheral nerves interact with skeletal muscles at the neuromuscular junction, modifications of this bidirectional communication by physical activity are positive to preserve this synapse as it increases quantal content and resistance to fatigue, acetylcholine receptors expansion, and myocytes’ fast-to-slow functional transition. Here, we provide the intermediate step between physical activity and functional and morphological changes by analyzing the molecular adaptations in the skeletal muscle of the full BDNF/TrkB downstream signaling pathway, directly involved in acetylcholine release and synapse maintenance. After 45 days of training at different intensities, the BDNF/TrkB molecular phenotype of trained muscles from male B6SJLF1/J mice undergo a fast-to-slow transition without affecting motor neuron size. We provide further knowledge to understand how exercise induces muscle molecular adaptations towards a slower phenotype, resistant to prolonged trains of stimulation or activity that can be useful as therapeutic tools

Adenosine Receptors in Developing and Adult Mouse Neuromuscular Junctions and Functional Links With Other Metabotropic Receptor Pathways

In the last few years, we have studied the presence and involvement in synaptogenesis and mature transmitter release of the adenosine autoreceptors (AR) in the mammalian neuromuscular junction (NMJ). Here, we review and bring together the previously published data to emphasize the relevance of these receptors for developmental axonal competition, synaptic loss and mature NMJ functional modulation. However, in addition to AR, activity-dependent mediators originating from any of the three cells that make the synapse (nerve, muscle, and glial cells) cross the extracellular cleft to generate signals in target metabotropic receptors. Thus, the integrated interpretation of the complementary function of all these receptors is needed. We previously studied, in the NMJ, the links of AR with mAChR and the neurotrophin receptor TrkB in the control of synapse elimination and transmitter release. We conclude that AR cooperate with these receptors through synergistic and antagonistic effects in the developmental synapse elimination process. In the adult NMJ, this cooperation is manifested so as that the functional integrity of a given receptor group depends on the other receptors operating normally (i.e., the functional integrity of mAChR depends on AR operating normally). These observations underlie the relevance of AR in the NMJ function

Brain-derived neurotrophic factor signaling in the neuromuscular junction during developmental axonal competition and synapse elimination

During the development of the nervous system, there is an overproduction of neurons and synapses. Hebbian competition between neighboring nerve endings and synapses performing different activity levels leads to their elimination or strengthening. We have extensively studied the involvement of the brain-derived neurotrophic factor-Tropomyosin-related kinase B receptor neurotrophic retrograde pathway, at the neuromuscular junction, in the axonal development and synapse elimination process versus the synapse consolidation. The purpose of this review is to describe the neurotrophic influence on developmental synapse elimination, in relation to other molecular pathways that we and others have found to regulate this process. In particular, we summarize our published results based on transmitter release analysis and axonal counts to show the different involvement of the presynaptic acetylcholine muscarinic autoreceptors, coupled to downstream serine-threonine protein kinases A and C (PKA and PKC) and voltage-gated calcium channels, at different nerve endings in developmental competition. The dynamic changes that occur simultaneously in several nerve terminals and synapses converge across a postsynaptic site, influence each other, and require careful studies to individualize the mechanisms of specific endings. We describe an activity-dependent balance (related to the extent of transmitter release) between the presynaptic muscarinic subtypes and the neurotrophin-mediated TrkB/p75NTR pathways that can influence the timing and fate of the competitive interactions between the different axon terminals. The downstream displacement of the PKA/PKC activity ratio to lower values, both in competing nerve terminals and at postsynaptic sites, plays a relevant role in controlling the elimination of supernumerary synapses. Finally, calcium entry through L- and P/Q- subtypes of voltage-gated calcium channels (both channels are present, together with the N-type channel in developing nerve terminals) contributes to reduce transmitter release and promote withdrawal of the most unfavorable nerve terminals during elimination (the weakest in acetylcholine release and those that have already become silent). The main findings contribute to a better understanding of punishment-rewarding interactions between nerve endings during development. Identifying the molecular targets and signaling pathways that allow synapse consolidation or withdrawal of synapses in different situations is important for potential therapies in neurodegenerative diseases

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

Abstract Background 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 M1 and M2 (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. Methods 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 M1 and M2 mAChRs, respectively. Diaphragm protein levels and phosphorylation’ changes were detected by Western blotting. Location of the target proteins was demonstrated using immunohistochemistry. Results 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, M1 and M2 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. Conclusion 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 holds the potential for identifying therapeutic strategies in conditions characterized by impaired neuromuscular communication