Utility of Animal Models for Studying the Human Neuromuscular Junction in Health and Disease

Abstract

The neuromuscular junction (NMJ) is a synapse formed between a lower motor neuron (LMN) and a skeletal muscle fibre. It comprises three major components: a motor nerve terminal, acetylcholine receptors (AChRs) at the motor endplate, and one or more terminal Schwann cells (tSCs). Structural and functional disruption of the NMJ is one of the earliest signs of neurodegeneration in motor neuron diseases (MND) such as amyotrophic lateral sclerosis (ALS), a fatal condition with no effective cure. ALS is characterized by progressive muscle weakness that eventually leads to paralysis and death from respiratory failure. Study of the NMJ is therefore critical to develop an understanding of both the disease process and the development of novel therapeutic interventions. For many decades, research has relied heavily on rodent models to study the NMJ. However, recent studies have revealed fundamental differences between rodents and humans in the cellular and molecular organization of the neuronal and muscular parts of the NMJ, as well as in the response to pathological events and therapeutic agents.

The first aim of this project was to extend our knowledge of human NMJ morphology by presenting the first detailed structural analysis of human tSCs. The findings demonstrated notable morphological differences between mouse and human tSCs. Despite having a similar number of tSCs per NMJ, human tSCs were significantly smaller in size and had more extended cytoplasm compared to murine tSCs. Furthermore, the positioning of the cell bodies relative to the endplate, which has clinical implications, was different between the two species: while mouse tSCs had significantly more synaptic nuclei, human nuclei were more widely distributed. These results provide further evidence for the stark morphological differences between mouse and human NMJs, encouraging the identification of alternative models that better represent the morphology of the human NMJ.

Based on these findings, the second part of the project explored NMJ morphology across several large mammalian species. The NMJs of the cat, dog, sheep, and pig were examined in a range of pelvic limb muscles, and baseline inter-species reference data were generated. These observations revealed that the morphology of sheep and pig NMJs more closely resembles that of the human NMJ compared with those of mice. As such, sheep and pigs may represent better models for translational work than either mice or rats.

Finally, to emphasize the critical role of the NMJ as an early target in MND, a thorough analysis of NMJ morphology was performed in an ALS mouse model (Thy1-hTDP-43). This part of the study aimed to characterize the morphological changes and the time course of denervation along with the differential vulnerability of NMJs in distinct body regions. Morphological assessment of NMJs was performed at three disease stages: pre-symptomatic, early symptomatic and end-stage; and in pelvic limb, cranial, thoracic limb muscles and abdominal muscles. Characteristically, muscle denervation was present in the pelvic limb from an early symptomatic stage. The disease progressed rapidly, and by the end-stage, all pelvic limb muscles showed severe denervation and loss of tSCs. In contrast, cranial muscles were only mildly denervated, whilst thoracic limb and abdominal muscles were remarkably preserved. Pathological features were mainly apparent in the pre-synaptic structures (nerve terminals and tSCs), whereas the post-synaptic structures (endplates and muscle fibres) were relatively unaffected. This comprehensive analysis and time course of NMJ pathology can be built-on to determine the sequence of pathological events occurring at the NMJ in ALS.

Overall, this project significantly deepens our understanding of differences between human NMJs and NMJs in other mammals, particularly mice. These differences must be considered when attempting to translate animal research to the human context. The project also emphasises the necessity of finding an appropriate animal model to accurately replicate the human NMJ in health and disease, encouraging further exploration of sheep and pig for future NMJ studies. And finally, it further emphasises the importance of the NMJ in MND and its early involvement in the pathogenesis of ALS.