The novel protein kinase C epsilon isoform at the adult neuromuscular synapse: location, regulation by synaptic activity-dependent muscle contraction through TrkB signaling and coupling to ACh release

Abstract

BackgroundProtein kinase C (PKC) regulates a variety of neural functions, including neurotransmitter release. Although various PKC isoforms can be expressed at the synaptic sites and specific cell distribution may contribute to their functional diversity, little is known about the isoform-specific functions of PKCs in neuromuscular synapse. The present study is designed to examine the location of the novel isoform nPKCε at the neuromuscular junction (NMJ), their synaptic activity-related expression changes, its regulation by muscle contraction, and their possible involvement in acetylcholine release.ResultsWe use immunohistochemistry and confocal microscopy to demonstrate that the novel isoform nPKCε is exclusively located in the motor nerve terminals of the adult rat NMJ. We also report that electrical stimulation of synaptic inputs to the skeletal muscle significantly increased the amount of nPKCε isoform as well as its phosphorylated form in the synaptic membrane, and muscle contraction is necessary for these nPKCε expression changes. The results also demonstrate that synaptic activity-induced muscle contraction promotes changes in presynaptic nPKCε through the brain-derived neurotrophic factor (BDNF)-mediated tyrosine kinase receptor B (TrkB) signaling. Moreover, nPKCε activity results in phosphorylation of the substrate MARCKS involved in actin cytoskeleton remodeling and related with neurotransmission. Finally, blocking nPKCε with a nPKCε-specific translocation inhibitor peptide (εV1-2) strongly reduces phorbol ester-induced ACh release potentiation, which further indicates that nPKCε is involved in neurotransmission.ConclusionsTogether, these results provide a mechanistic insight into how synaptic activity-induced muscle contraction could regulate the presynaptic action of the nPKCε isoform and suggest that muscle contraction is an important regulatory step in TrkB signaling at the NMJ.

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

“…Thus, they are ideal targets for both presynaptic and retrograde neurotrophic control induced by muscle contraction activity. Presynaptic activity decreases nPKCε and cPKCβI by an activation-induced degradation mechanism and muscle contraction increases its protein levels and both mechanisms are linked to the BDNF/TrkB activity [36,[84][85][86]. This evidences the cross-linking between the pre and postsynaptic activities and the neurotrophic retrograde control to maintain a stable and sufficient pool of these PKC isoforms at the nerve terminal.…”

“…Depending on the isoform, they require Ca 2+ and/or diacylglycerol and phosphatidyl serine. Two PKC isoforms, nPKCε and cPKCβI, are exclusively presynaptic at the NMJ and participate in regulating ACh release [36,[84][85][86]. Thus, they are ideal targets for both presynaptic and retrograde neurotrophic control induced by muscle contraction activity.…”

“…This has a direct and unequal impact in PKC isoforms' phosphorylation and activity. Several PKC isoforms located at the presynaptic and postsynaptic components that regulate neurotransmission at the healthy NMJ [36,[84][85][86]130,131] are differently affected in ALS muscles [95,132] (Figure 2b), probably due to their different mechanisms of activation. Firstly, the major activation of nPKCθ is linked with the inhibition of its target ClC-1 chloride channel [132] that results in morphological alterations of the neuromuscular presynaptic terminals, a high turnover rate of AChR and NMJ dismantlement in ALS mice [133].…”

Synaptic Activity and Muscle Contraction Increases PDK1 and PKCβI Phosphorylation in the Presynaptic Membrane of the Neuromuscular Junction

“…Protein kinase C (PKC) is a common signaling node of many cellular processes, being a crucial regulator of neuronal excitability, neurotransmitter release and synaptic transmission in the nervous system (Dempsey et al, 2000 ; Lanuza et al, 2014 ; Tomàs et al, 2014 ). Several PKC isoforms are expressed and differently regulated in the skeletal muscle and, particularly, at the neuromuscular junction (NMJ; Hilgenberg and Miles, 1995 ; Lanuza et al, 2000 ; Perkins et al, 2001 ; Li et al, 2004 ; Besalduch et al, 2010 , 2013 ; Obis et al, 2015a , b ). Specifically, the conventional PKC βI (cPKCβI) has been involved in the regulation of diverse cellular functions including neurotransmission (Hurtado et al, 2017 ).…”

Protein kinase C ϵ stabilizes β-catenin and regulates its subcellular localization in podocytes

“…PKC⑀ belongs to the subfamily of novel PKCs and is activated via DAG, different from conventional isoforms, which are activated by calcium and DAG. PKC⑀ was shown previously to play an essential role in cell proliferation, migration, invasion, and survival, and lack of PKC⑀ is linked to reduced cardioprotective effects (6) and neurotransmission (7). Moreover, PKC⑀ has been well characterized as an important binding protein of actin and, thus, has a regulatory function for the cytoskeleton (8,9).…”