Muscle Contraction Regulates BDNF/TrkB Signaling to Modulate Synaptic Function through Presynaptic cPKCα and cPKCβI.

Erica Hurtado,Josep Tomàs,Maria A Lanuza,Anna Simó,Víctor Cilleros,Laura Nadal,Cynthia L Jordan,Teresa Obis,Katherine Halievski,Manel M Santafé,Marta Tomàs,Neus Garcia

doi:10.3389/fnmol.2017.00147

Abstract

The neurotrophin brain-derived neurotrophic factor (BDNF) acts via tropomyosin-related kinase B receptor (TrkB) to regulate synapse maintenance and function in the neuromuscular system. The potentiation of acetylcholine (ACh) release by BDNF requires TrkB phosphorylation and Protein Kinase C (PKC) activation. BDNF is secreted in an activity-dependent manner but it is not known if pre- and/or postsynaptic activities enhance BDNF expression in vivo at the neuromuscular junction (NMJ). Here, we investigated whether nerve and muscle cell activities regulate presynaptic conventional PKC (cPKCα and βI) via BDNF/TrkB signaling to modulate synaptic strength at the NMJ. To differentiate the effects of presynaptic activity from that of muscle contraction, we stimulated the phrenic nerve of rat diaphragms (1 Hz, 30 min) with or without contraction (abolished by μ-conotoxin GIIIB). Then, we performed ELISA, Western blotting, qRT-PCR, immunofluorescence and electrophysiological techniques. We found that nerve-induced muscle contraction: (1) increases the levels of mature BDNF protein without affecting pro-BDNF protein or BDNF mRNA levels; (2) downregulates TrkB.T1 without affecting TrkB.FL or p75 neurotrophin receptor (p75) levels; (3) increases presynaptic cPKCα and cPKCβI protein level through TrkB signaling; and (4) enhances phosphorylation of cPKCα and cPKCβI. Furthermore, we demonstrate that cPKCβI, which is exclusively located in the motor nerve terminals, increases activity-induced acetylcholine release. Together, these results show that nerve-induced muscle contraction is a key regulator of BDNF/TrkB signaling pathway, retrogradely activating presynaptic cPKC isoforms (in particular cPKCβI) to modulate synaptic function. These results indicate that a decrease in neuromuscular activity, as occurs in several neuromuscular disorders, could affect the BDNF/TrkB/PKC pathway that links pre- and postsynaptic activity to maintain neuromuscular function.

Highlights

Nerves and skeletal muscles interact via two modes of communication: electrical activity and neurotrophic regulation (Baldwin et al, 2013; Cisterna et al, 2014)
To determine the relationship between neuromuscular activity and neurotrophic control, we developed an in vivo experimental system in which we can distinguish the effects of synaptic activity from that of muscle contraction
When we examine the expression of cPKCβI based on immunofluorescent labeling of semithin cross-sections of levator auris longus (LAL) muscles (Figure 6A), we find cPKCβI fine granular green immunofluorescence located over the postsynaptic line of the nicotinic acetylcholine receptor site and colocalized with the neurofilament and syntaxin (NF+Synt, in blue top image 1) in the nerve terminal. cPKCβI is not colocalized with labels for either Schwann cells (S100, in blue bottom image 2) or Acetylcholine receptors (AChRs) postsynaptically

Summary

Introduction

Nerves and skeletal muscles interact via two modes of communication: electrical activity and neurotrophic regulation (Baldwin et al, 2013; Cisterna et al, 2014). Neurotrophic control acts via the release of neurotrophic factors, including the neurotrophins, and regulates the development, differentiation, survival and function of the nerve terminal (Wang et al, 1995; Mantilla et al, 2004). One of the most studied neurotrophins is brain-derived neurotrophic factor (BDNF; Hofer and Barde, 1988; Barde, 1990; Bibel and Barde, 2000). TrkB.T1 is the main truncated isoform in the skeletal muscle, being TrkB.T2 a variant more predominant in the brain tissue (Stoilov et al, 2002). Evidence suggests that heterodimers of TrkB.FL with the truncated isoforms inhibit trans-autophosphorylation of TrkB.FL, reduce BDNF signaling or even may signal independently (Eide et al, 1996; Baxter et al, 1997; Rose et al, 2003; Dorsey et al, 2012; Wong and Garner, 2012)

Methods

Results

Conclusion