Year

2018

Degree Name

Doctor of Philosophy

Department

School of Chemistry

Abstract

Contractile muscle activity is controlled by the highly reliable motor neuron-muscle system. The regulation of this system involves the transmission of action potentials from the central nervous system to muscle fibres at neuromuscular junctions (NMJs). This complex system relies on dynamic interactions of cell adhesion and signalling molecules and cell membrane proteins to release neurotransmitters from motor neurons into the synaptic cleft, followed by neurotransmitter binding to specific receptors: acetylcholine receptors (AChR), that are located at the end plate of the plasma membrane of muscle fibres. There are many factors to be considered when investigating NMJ formation, maturation and fast and reliable synaptic transmission, however, recent data reveals that clustering and maintenance of high densities of AChR are key elements of synaptogenesis at the NMJ.

Conventionally, it was believed that nerve dictates NMJ formation; this concept was adopted due to the fact that nerve sprouts appeared to determine the location of synapses on the sarcolemma during reinnervation of denervated muscle. However, this conventional view has been challenged by more recent theories such as the myocentric model of synaptogenesis and the dying-back hypothesis. The myocentric model states that muscles have an intrinsic capacity to regulate NMJ formation independent of neural signaling. In fact, acetylcholine receptors (AChR), a critical component of the postsynaptic apparatus, develop on muscles before the arrival of neurites during early embryonic development. It is believed that these “aneural” clusters of AChR on muscles restrict the incoming nerve and induce synaptogenesis within a predetermined territory on the sarcolemma. Furthermore, the alternative view “dying-back” has growing evidence which supports the idea that dysfunction of these junctions may play a key role in several neuromuscular diseases. For example, there is growing evidence supporting the hypothesis that the survival of NMJs is essential to delay the progression of ALS.

From a therapeutic perspective the myocentric model and the die back hypothesis raise the possibility of new therapies where increasing aneural cluster formation on muscle, as well as the stabilization of NMJs through artificial treatments, are promising approaches to attenuate the progression of muscle wasting disorders, making NMJs a good indicator of motor neuron health.

Current literature reviews suggest that during development neurons target and form synapses driven by dynamic interactions of biophysical and biochemical cues, whilst electrical activity, in the form of ion transients, plays a role in neuronal development both before and after synapse formation. In addition, it has recently been shown that the formation and architecture of NMJs can be influenced by electrical stimulation (ES) in vitro, however, the mechanisms underlying the response of the neuromuscular complex to this form of stimulation is not fully understood. Many in vitro and in vivo studies have been conducted using external ES to control cell characteristics, indicating that ES has positive benefits in many areas such as woundhealing, bone growth, pain relief, muscle restoration, proliferation and differentiation of stem cells, as well as in nerve guidance and growth. It is clear from this that ES of cells and tissues is a potential therapeutic tool for many neural, muscular and other disorders. The exact parameters of ES applied, including stimulation type, current or voltage density, direct or field stimulation, and frequency differ widely in the literature, and detailed studies on which ES parameters are safe and effective to achieve desired outcomes for different tissue types have not been conducted.

This thesis attempts to analyse the effect of ES on AChR clusters available for NMJ formation. Additionally, the project attempts to establish and optimize the parameters for this ES in a two dimensional (2D) environment. Furthermore, the work aims to explore techniques and models to replicate this in vitro co-culture system in three dimensions (3D). Different biomaterials (collagen, gellan gum-GG and GG-RGD) were used to encapsulate and grow nerve and muscle cells (both primary and cell lines) in 3D. While this encapsulation method allowed cells to grow in a 3D fashion, the technique still has some limitations when trying to place cells in a precise location or build composites with complex architectures. 3D printing technology has shown to be a promising tool to address some of these limitations via the precise placement of cells within hydrogels in a layer-by-layer fashion. Here a versatile, 3D printing technique was introduced where primary cortical neurons were 3D printed in a layered fashion. This printing process offered the opportunity to develop more accurate in vitro brain models (“brain in a bench top”) that could be applied to study neuronal cell behavior. The brain model could also provide a tool for assessing the efficacy and gaining insights into the mode of action of new therapies, as well as providing a better understanding of brain injuries and neurodegenerative diseases.

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