The Role of Activity in Neuromuscular Synaptic Degeneration: Insights from Wld

The nervous system is a dynamic structure. Both during development and in the adult, synapses display activity-dependent plasticity which can modify their structure and function. In the neonate, activity influences the stability of functional connections between the muscle and nerve. In adults, the process of neurotransmitter release and the structure of the postsynaptic muscle can also be altered by external stimuli such as exercise. It is important to understand this plasticity of the neuromuscular system, the ways in which it can be modified, and its relationship to the maintenance or degeneration of synapses. After injury, peripheral nerve undergoes Wallerian Degeneration, during which the connections between axons and muscle fibres are lost, followed by the fragmentation of the nerve itself. The primary goal of this thesis was to determine whether activity modulates this process; that is, whether enhancing or reducing neuromuscular activity creates a susceptibility to degeneration or alternatively provides any protection against it. Developing greater understanding of this process is essential in relation to neurodegenerative disorders in which the benefits of activity, in the form of exercise, are controversial. Using Wld s mice, in which synaptic degeneration occurs approximately ten times more slowly than normal after nerve injury, I investigated the influence of both decreased (tetrodotoxin induced paralysis) and increased (voluntary wheel running) activity in vivo on this process. Paralysis prior to axotomy resulted in a significant increase in the rate of synapse degeneration. Using a novel method of repeatedly visualising degenerating synapses and axons in vivo I also established that this effect was specific to the synapse, as it did not affect the degeneration of axons. In contrast, voluntary wheel running had no effect on the rate of either axonal or neuromuscular synapse degeneration, but induced a slight modification of neuromuscular transmission. To provide a more stringent test I developed a novel assay based on overnight, ex vivo incubation of nerve-muscle preparations at 32°C. I first demonstrated that this procedure separates the different degeneration time courses for neuromuscular synapse degeneration in wild-type and Wld s preparations. I then extended the study to investigate further ways of modulating synaptic degeneration. First, I tested the effects of electrical stimulation. Intermittent high frequency (100Hz) stimulation reduced the level of protection. Finally, I tested the effects of NAMPT enzymatic inhibitor FK866 on synaptic degeneration. Interestingly, the synaptic protection observed in Wld s muscles was enhanced in the presence of FK866. The results of my findings are relevant to understanding the plasticity of synapses and its relationship to degeneration. Together, these studies highlight the potential of genetic and epigenetic factors, including activity, to regulate neuromuscular synapse degeneration. My study also provides proof of concept for a novel organotypic culture system in which to identify pharmacological modulators of synaptic degeneration that could form part of a second-line screen for neuroprotective compounds or phenotypes. My findings may be viewed in the wide context of neurodegenerative disease, since synaptic use or disuse is widely thought to influence susceptibility, onset and progression in such disorders.