Antibody to MyoD Decreases Myogenin Gene Expression and ... iThflThffftα μftαTh a ciechnol ournal...

a SciTechnol journal Research Article Anderson et al, J Neurosci Clin Res 2017, 2:1 Journal of Neuroscience & Clinical Research All articles published in Journal of Neuroscience & Clinical Research are the property of SciTechnol, and is protected by copyright laws. Copyright © 2017, SciTechnol, All Rights Reserved. International Publisher of Science, Technology and Medicine Antibody to MyoD Decreases Myogenin Gene Expression and Agrin-induced Acetylcholine Receptor Clustering Erica E Anderson 1 , David H Campbell 2 , Kelly Ezell 1 , Paul R Standley 2 and Wade A Grow 1 * Abstract Objective: A family of myogenic regulatory factors, including MyoD and myogenin, guide myogenesis and neuromuscular synapse formation. Myogenin gene expression is activated by MyoD, and one of myogenin’s functions is to activate gene expression of the acetylcholine receptor (AChR) at the neuromuscular synapse. Motor neurons release agrin as they near skeletal muscle fibers in development, which drives the clustering of existing AChRs to the site of neuromuscular synapse formation. We have previously demonstrated that continuous exposure to antibody to MyoD or myogenin decreases agrin-induced AChR clustering in C2C12 skeletal muscle cell culture. Our objective was to more specifically establish how MyoD and myogenin interact in development. Methods: C2C12 cell cultures were exposed to experimental manipulations including antibodies to MyoD and myogenin, and myogenin morpholino. Endo-Porter was used to enhance cell uptake of experimental manipulations. AChR clustering assays were performed to assess the effect of antibody or morpholino on agrin-induced AChR clustering. Western blots were performed to assess myogenin gene expression after antibody or morpholino exposure. Results: The results reported here demonstrate that exposure as short as eight hours for antibody to myogenin can decrease agrin-induced AChR clustering in myotubes. We have previously demonstrated that some experimental manipulations reduce myogenin gene expression concurrent with a decrease in agrin- induced AChR clustering. The current results establish more specifically how MyoD and myogenin interact in neuromuscular synapse formation by demonstrating that exposure to antibody to MyoD reduces myogenin gene expression concurrent with a decrease in agrin-induced AChR clustering. Conclusion: These results suggest that MyoD is essential for agrin-induced AChR clustering through a mechanism that includes activation of myogenin gene expression, leading to activation of AChR gene expression, and ultimately production of an appropriate level of AChR for agrin-induced AChR clustering and neuromuscular synapse formation. *Corresponding author: Wade A Grow, Department of Anatomy, Arizona College of Osteopathic Medicine, Midwestern University, 19555 N. 59th Avenue, Glendale, AZ 85308, USA, Tel: 623-572-3686; Fax: 623-572-3679; E-mail: [email protected] Received: December 31, 2016 Accepted: February 10, 2017 Published: February 16, 2017 Introduction Myogenic regulatory factors MyoD (myf3), myogenin (myf4), myf5, and MRF4 (myf6) guide skeletal muscle development. ey evolved from a single gene as a result of gene duplication events and subsequent mutations [1]. ese basic helix-loop-helix transcription factors bind to the E-box found in the promoters or enhancers of many muscle-specific genes to activate or inhibit transcription [2-6]. Western blots using C2C12 cell culture verified in vitro the temporal expression pattern initially determined with experiments using knockout mice. MyoD was expressed in proliferating myoblasts and myotubes, while myogenin was primarily expressed in myotubes [7]. is expression pattern, where MyoD is expressed early in myogenesis and myogenin is expressed later in myogenesis, was also demonstrated in C2C12 cell culture using immunofluorescence [8]. Skeletal muscle cell cultures, such as the C2C12 cell line derived from mouse hindlimb, provide simplified systems for studying myogenesis and more specifically the development of the postsynaptic component of the neuromuscular synapse [9,10]. is postsynaptic component includes acetycholine receptors (AChRs), as well as other molecules that aggregate and co-localize with AChR, including a low-density lipoprotein receptor-related protein (Lrp4), a muscle-specific kinase (MuSK), and rapsyn. Activated motor neurons release acetylcholine, which binds AChRs as a first step in muscle fiber contraction. In early development AChRs cluster spontaneously but aggregation increases upon exposure to motor neuron derived agrin [11-13]. As motor neurons approach skeletal muscle fibers in development, agrin is released and binds to Lrp4, forming a complex of Lrp4 and MuSK which mediates MuSK activation by agrin [14-16]. Agrin binding stimulates tyrosine phosphorylation of MuSK [17] and Lrp4 [15], which drives a subsequent signaling pathway that includes tyrosine phosphorylation of AChRs leading to increased AChR clustering [18,19]. MuSK and rapsyn are essential for AChR clustering during neuromuscular synapse formation [17,20], with MuSK required for the signaling events that precede AChR clustering [17,21-23]. In addition, AChRs are required for the agrin- induced aggregation of MuSK at the neuromuscular synapse [24]. Suppression of Lrp4 gene expression decreases agrin binding activity, agrin-induced MuSK tyrosine phosphorylation, and agrin-induced AChR clustering [15]. In myogenesis, the expression of early genes is initiated by MyoD, while later expression of other genes is initiated by MyoD and myogenin. In the absence of MyoD, myogenin can inefficiently activate early genes [25]. In addition to increasing myogenin gene expression, MyoD also targets MuSK and rapsyn gene expression, while myogenin targets rapsyn but not MuSK gene expression [7]. Moreover, myogenin activates genes for AChR subunits [26,27]. Overexpression of myogenin in transgenic mice elevates mRNA and protein levels of all five AChR subunits, while reducing MyoD protein levels [28]. is suggests that myogenic regulatory factors Keywords AChR; Agrin, C2C12; Gene expression; MyoD; Myogenin; Neuromuscular synapse

Antibody to MyoD Decreases Myogenin Gene Expression and ... iThflThffftα μftαTh a ciechnol ournal...

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