Electrical stimulation accelerates neuromuscular junction formation through ADAM19/neuregulin/ErbB signaling in vitro

Abstract

The mechanism by which electrical stimulation affects formation of neuromuscular junctions (NMJs) remains unknown. NG108-15, a neural cell line, is commonly used in in vitro co-culture models of myotubes to observe synapse formation; therefore, we employed this model to observe the effects of electrical stimulation on NMJ formation. Initially, L6 cells were differentiated and NG108-15 cells were then added to the same culture dish. After 2 and 3 days of co-culture, the cells were electrically stimulated at 50 V and 0.5 Hz for 0, 5, 30, and 60 min (C, ES5, ES30, and ES60 groups, respectively) and were analyzed after co-culture for 4 days. Immunofluorescence experiments showed significantly increased aggregation of acetylcholine receptors and inhibition of neural outgrowth in the ES30 and ES60 groups. Furthermore, ADAM19 and phospho-ErbB3 were found to be specifically localized in co-cultured NG108-15 cells. Immunoblotting demonstrated that synapsin 1, ADAM19 precursor and its activated form, phospho-ErbB3, and ERK1 protein levels had increased in an electrical stimulation period-dependent manner. Thus, we found that electrical stimulation accelerated NMJ formation, possibly through activation of ADAM19/neuregulin/ErbB signaling in NG108-15 cells.

Fabrication of a Biocompatible Liquid Crystal Graphene Oxide–Gold Nanorods Electro‐ and Photoactive Interface for Cell Stimulation

“…Hence, NG108‐15 cells mostly adopted a bipolar or multipolar morphology. This denotes normal differentiation of the cell line on LCGO–AuNR films as NG108‐15 cells are models for development and differentiation of cholinergic peripheral neurons (including motor neurons), which have this morphology . Moreover, the proportion of cells expressing one, two, three, or more neurites on Day 3 ( n = 434) and Day 7 ( n = 484) on LCGO–AuNR films were statistically different compared to the controls (Pearson Chi‐square; p ≤ 0.05), with LCGO–AuNR films showing a higher proportion of cells expressing two or more neurites (85%) on Day 7 (Figure c).…”

Creating Interactions between Tissue-Engineered Skeletal Muscle and the Peripheral Nervous System

“…It has been shown that repetitive electrical stimulation of motor neuron-myotube cultures for 30–60 min significantly up-regulates synapsin expression and AChR clustering, suggesting such stimuli may be employed to improve NMJ formation in culture [Fukazawa et al, 2013]. This result is logical, given the activity dependence of NMJ structure and development in vivo [Ataman et al, 2008].…”

Identification of tool use acquisition‐associated genes in the primate neocortex

“…ADAM19 is a member of the 'A Disintegrin And Metalloprotease' (ADAM) family that modulates various signaling pathways by cleaving and shedding membrane-bound growth factors or their receptors (Alfandari et al 2009;Reiss & Saftig 2009). ADAM19 is involved in cardiovascular morphogenesis and neural and muscle development by cleavage of HB-EGF, cadherin, or neuregulin-1 (Shirakabe et al 2001;Zhou et al 2004;Komatsu et al 2007;Neuner et al 2008;Fukazawa et al 2013;Schiffmacher et al 2014).…”