Cholinergic neurons regulate secretion of glial cell line-derived neurotrophic factor by skeletal muscle cells in culture

Abstract

Glial cell line-derived neurotrophic factor (GDNF) has been identified as a potent survival factor for both central and peripheral neurons. GDNF has been shown to be a potent survival factor for motor neurons during programmed cell death and continuous treatment with GDNF maintains hyperinnervation of skeletal muscle in adulthood. However, little is known about factors regulating normal production of endogenous GDNF in skeletal muscle. This study aimed to examine the role that motor neurons play in regulating GDNF secretion by skeletal muscle. A co-culture of skeletal muscle cells (C2C12) and cholinergic neurons, glioma × neuroblastoma hybrid cells (NG108-15) were used to create nerve muscle interactions in vitro. Acetylcholine receptors (AChRs) on nerve-myotube co-cultures were blocked with alpha bungarotoxin (α-BTX). GDNF protein content in cells and in culture medium was analyzed by enzyme-linked immunosorbant assay (ELISA) and western blotting. GDNF localization was examined by immunocytochemistry. The nerve-muscle co-culture study indicated that the addition of motor neurons to skeletal muscle cells reduced the secretion of GDNF by skeletal muscle. The results also showed that blocking AChRs with α-BTX reversed the action of neural cells on GDNF secretion by skeletal muscle. Although ELISA results showed no GDNF in differentiated NG108-15 cells grown alone, immunocytochemical analysis showed that GDNF was localized in NG108-15 cells co-cultured with C2C12 myotubes. These results suggest that motor neurons may be regulating their own supply of GDNF secreted by skeletal muscle and that activation of AChRs may be involved in this process.

Effects of acetylcholine and electrical stimulation on glial cell line-derived neurotrophic factor production in skeletal muscle cells

“…The inhibition of GDNF production caused by short-term depolarization may explain results of previous studies which show that GDNF production is inhibited when skeletal muscle is co-cultured with cholinergic neurons (Vianney and Spitsbergen, 2011). The inhibitory effect of ACh or short-term electrical stimulation may also help explain why GDNF mRNA levels increase in skeletal muscle following denervation (Lie and Weis, 1998), and why ACh inhibits extra cholinergic nerve branching during development (An et al 2010).…”

“…In previous studies we showed that cholinergic neurons play a role in regulating GDNF synthesis and release by skeletal muscle (Vianney and Spitsbergen, 2011). Here, we sought to determine whether cholinergic neurons exert their effect via neurotransmitter release.…”

“…The C2C12 cell line was extracted from 2-month old mouse thigh muscle and the cells have been used by researchers because of their in vivo skeletal muscle phenotype, including expression of contractile proteins (Ling et al, 2005; Yafel and Saxel, 1977). Culturing procedures were performed according to the ATCC protocols and as described by Vianney and Spitsbergen (2011), with some minor modifications. Briefly, C2C12 myoblasts were initially seeded on 100-mm plates and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM: ATCC) supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic (Life Technologies, Carlsbad, CA, USA).…”

“…Dennervation of skeletal muscle causes an increase in GDNF expression (Suzuki et al, 1998b; Lie and Weis, 1998), while muscle cells co-cultured with neural cells in vitro secrete less GDNF (Vianney and Spitsbergen, 2011). These findings suggest that the innervation status of skeletal muscles plays a role in regulating the amount of GDNF produced by muscle.…”

“…These findings suggest that the innervation status of skeletal muscles plays a role in regulating the amount of GDNF produced by muscle. In cell culture, the ratio of GDNF inside skeletal muscle is higher than that released into culture medium (Vianney and Spitsbergen, 2011), suggesting that GDNF may be synthesized and stored in a manner similar to neurotrophins (Poo et al, 2001). In vivo studies have shown that GDNF in skeletal muscle can be regulated in an activity-dependent manner, such as with physical exercise (McCullough et al, 2011; Wehrwein et al, 2002).…”