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

Abstract

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.

The p75NTR neurotrophin receptor is required to organize the mature neuromuscular synapse by regulating synaptic vesicle availability

“…For instance, hind limb muscles of amyotrophic lateral sclerosis mouse models display a significant decline in the number of NMJs expressing BDNF, NT-4, GDNF, p75 NTR , TrkB, and TrkC [31]. Therefore, as a first hint to analyze the potential involvement of NT-dependent signaling on the morphological and functional alterations observed in p75 NTR −/− mice, we analyzed the levels of expression of BDNF and its TrkB receptor at the NMJ, as BDNF/TrkB signaling plays important roles on NMJ organization, including synaptic vesicle availability [34, 75]. With that aim, cryosections of TA muscles from control and p75 NTR −/− mice were subjected to immunohistochemistry to detect BDNF and an active form of TrkB (p-TrkB Y816) (Fig.…”

The Impact of Kinases in Amyotrophic Lateral Sclerosis at the Neuromuscular Synapse: Insights into BDNF/TrkB and PKC Signaling

“…In the peripheral nervous system (PNS), TrkB has been implicated in the maintenance of the synaptic function and the structural integrity in adult muscular synapses [42]. Moreover, TrkB signaling also plays an important role in the neuromuscular bidirectional communication, as both the synaptic activity and the postsynaptic muscle contraction potentiate its action to activate presynaptic kinases on demand to control ACh release [36,39,40] (Figure 2a).…”

“…A molecular approach recently demonstrated that several key proteins of the exocytotic mechanism of the synaptic vesicles at the NMJ are targets of nPKCε and cPKCβI [39,40]. In brief, the activity-induced phosphorylation of Munc18-1, a regulatory molecule of the exocytotic apparatus in the nerve terminals, depends on the coordinated action of both kinases and involves retrograde BDNF/TrkB signaling [39]. Moreover, SNAP-25, a member of the soluble N-ethyl maleimide sensitive-factor attachment protein receptors (SNARE) complex, is only phosphorylated by nPKCε independently of the neurotrophic control [40].…”

“…Moreover, we widely investigated the functional complex network constituted by these receptors and the presynaptic serine-threonine kinases, PKC and PKA, to regulate ACh release at the NMJ [38]. Furthermore, we presented the individual and complementary actions of presynaptic activity and the resulting muscle contraction over presynaptic kinases, and showed that BDNF/TrkB signaling is key in their regulation [36,39,40], which reveals the mutual intercommunication and regulation between the nerve terminal and the myocyte activities.…”

Myasthenia Gravis: From the Viewpoint of Pathogenicity Focusing on Acetylcholine Receptor Clustering, Trans-Synaptic Homeostasis and Synaptic Stability

“…This may reflect the impairment of the presynaptic Ca 2+ homeostasis for ACh release (Takamori, 2012) to compensate for postsynaptic dysfunction. The presynaptic Ca 2+ homeostasis is promoted by the activation of G protein-coupled receptors such as M1-type muscarinic AChR (M1 mAChR) and A2A adenosine receptor along with the interaction of brain-derived neurotrophic factor (BDNF) with receptor tyrosine kinase B (TrkB); these biological mechanisms lead to P/Q-type VGCC activation by the signaling mediated by phospholipase C (PLC)-phosphatidylinositol 4,5-bisphosphate (PIP2)-diacylglycerol (DAG)-protein kinase C (PKC; Amaral and Pozzo-Miller, 2012;Santafé et al, 2015;Nadal et al, 2016;Hurtado et al, 2017;Simö et al, 2018;Tomàs et al, 2018). Also, PLC-generated DAG regulates the presynaptic vesicle priming protein (Munc13-1) to recruit ACh-containing vesicles for the immediately releasable pool (Bauer et al, 2007).…”