Investigating factors that can influence the rate of neuromuscular junction degeneration in injury and disease
Mole, Alannah Jane
Axonal and synaptic degeneration occur in response to nerve injury and during neurodegenerative disease. Traumatic nerve injury leads to rapid fragmentation of the distal axon and loss of synaptic terminals in a process known as Wallerian degeneration (WD). This is morphologically distinct from some motor neuron diseases, like Spinal Muscular Atrophy (SMA), where axons gradually retract, or die-back, from the motor endplate. Identifying and understanding factors that can influence the rate of degeneration is of significant clinical and biological importance, to facilitate our knowledge of the mechanisms of neurodegeneration and to aid in the identification of novel therapeutic targets. Factors that can change the speed of axonal and synaptic breakdown are interesting, as targeting these could represent an approach to slow or prevent breakdown in disease. The work contained within this thesis aims to identify factors that can alter the rate of synaptic degeneration in morphologically distinct types of synaptic degeneration, including following injury, during disease and during postnatal development. Using an ex vivo model of peripheral nerve injury, it is apparent that synaptic withdrawal is slower during early postnatal development, and that this delay is progressively lost over time. For example, significantly more neuromuscular junctions (NMJs) remain fully occupied in the cranial muscle preparations at postnatal day (P) 15 than P25. Intermuscular variability was also found to be a feature of WD, with less synaptic retraction evident in the abdominal preparations compared to cranial muscles. Importantly, differences in synaptic responses to injury were not consistent with patterns of neuromuscular vulnerability that have been previously described in mouse models of SMA, which is caused by a lack of survival motor neuron protein (Smn). To further investigate the relationship between SMA and WD, nerve injury was induced in preparations from the Smn2B/- mouse model of SMA. In a disease-resistant muscle (rostral band of levator auris longus), where there is minimal denervation, the level of synaptic loss in response to injury is similar to wild-type, suggesting that loss of Smn alone is not sufficient to influence rates of WD, suggesting that WD is Smn-independent. However, in a disease-vulnerable muscle with ongoing degeneration (transversus abdominis), the level of synaptic loss in response to injury is significantly increased, with the percentage of denervated endplates increasing by 33% following injury. Thus, the presence of dying-back pathology appears to accelerate synaptic loss. Next, analysis of two independent proteomic datasets highlighted that there are changes in mitochondrial proteins during postnatal development that correlate with increases in synaptic vulnerability following injury during this time frame. Expanding this analysis to assess mitochondrial-associated protein levels and mitochondrial DNA levels in a range of different muscles revealed that although there are global changes in mitochondrial proteins across development, complex I levels do not correlate with differences in the synaptic response to injury. There is evidence, however, that increases in complex I in nerve may underlie intermuscular differences. The final part of this work was to determine whether reductions in P53, a factor that is involved in cell death processes, could alter the rate of synaptic loss under different scenarios. Application of the ex vivo model, and immunohistochemical and transcriptional analysis in an inducible P53-knockout mouse model demonstrated that postnatal reduction in P53 has no effect on the rate of synaptic loss following injury, during a die-back, or during development, suggesting P53-independence. Overall, the expanded ex vivo model and workflows described here represent powerful tools with which to study factors or pathways that can influence the rate of synaptic degeneration under different physiological scenarios. This will provide insight into mechanistic commonalities in synaptic degeneration that could ultimately facilitate the identification of a common therapeutic target in a range of neurodegenerative conditions.