Cellular and molecular anatomy of the mammalian neuromuscular junction in health and disease
The vast diversity across the animal kingdom and in particular within the class of mammals is not only of evolutionary interest, but also plays an important role in framing the context of research in which mammalian animal models are utilised. Research addressing neurodegenerative diseases has struggled to translate from the widely used mouse model to humans. We now know the neuromuscular junction (NMJ) - the key player responsible for generation of movement and often a target in disease - is strikingly different between mouse and human, thus questioning the validity of rodent models. Building upon recent studies assessing the healthy human NMJ, this project directly sought to examine the human NMJ in disease and whether predictions made from mouse models also applied in humans. Furthermore, this project aimed to identify mammalian models more similar to human NMJ morphology, which could be used as alternatives to the mouse. Following morphological NMJ analysis of the human and mammalian NMJ, molecular pathways contributing to the stability of the human NMJ during healthy ageing of muscle were analysed, as this knowledge could prove beneficial in the generation of more translatable animal models and therapies. To evaluate whether changes at the NMJ as predicted from mouse models also occurred in humans with muscle wasting, NMJ morphology was compared between patients with cancer cachexia, weight stable cancer patients, and control patients. Surgical muscle biopsies were sampled, NMJs were immunofluorescently visualised, imaged and analysed using ‘aNMJ-morph’, a semi-automated macro version built upon the well-established workflow ‘NMJ-morph’. Whilst cancer cachectic patients showed clear signs of muscle atrophy, NMJ morphology was found to be stable with no signs of denervation. This suggests that current mouse models of cancer cachexia, modelling denervation, should be translated with caution. Given the stark differences between mouse and human NMJs in health and disease, the development of more suitable animal models is fundamental. Therefore, exploration of NMJ morphology across other mammalian species (cat, dog, sheep, pig, and pony) in comparison to the human was required. Overall, sheep and pig proved to be the most similar to human NMJ morphology across pelvic/lower limb muscles, making them attractive models to explore further. Whilst it is advantageous to explore other mammalian models, it is ultimately critical to better understand the human NMJ in health and disease in order to mimic disease appropriately, regardless of model. The ageing related loss of muscle mass and function, sarcopenia, is a major public health problem and it is thus important to evaluate molecular pathways that occur during healthy ageing at the level of both muscle and the NMJ. Whilst much is known about the ageing of human muscle, the molecular pathways governing healthy ageing at the NMJ itself remain unexplored. This project shows that in comparison to “Middle aged” subjects (40 yo –50 yo), the muscle (devoid of the NMJ) of “Senior” (60 yo) and “Old” (70 yo –80 yo) subjects is in an insulin resistant state, with dysregulation of pathways associated with oxidative phosphorylation and energy production. NMJ-enriched samples of Senior and Old subjects are more similar to Middle aged subjects in protein abundance, and pathways associated with insulin sensitivity are upregulated. Overall, this project emphasizes the stark morphological difference between NMJs of the mouse and the human, or other mammals. In particular the stability of the human NMJ during muscle wasting diseases such as cancer cachexia, or on a molecular level, during ageing, is striking. Therefore, there is an unmet need to find animal models that are more suitable to model human diseases. This project found that the sheep and pig are suitable to be explored as models of human neuromuscular diseases, opening up avenues of research towards more translatable larger mammalian models.