The neuromuscular junction (NMJ), the synapse formed between lower motor neuron and skeletal muscle fibre, is known to be a target in a number of neurodegenerative conditions, including motor neuron disease (MND). Located in an accessible part of the peripheral nervous system, the NMJ can be used as a ‘model synapse’ in the context of ‘connectomics’ – the study of synaptic connectivity throughout the nervous system as a whole. Although the NMJ has been studied in a number of species, relatively little is known about its structure in humans, complicating the translation of animal models of disease to the human condition. Described here is the first detailed cellular and molecular characterization of the human NMJ. A standardized methodology for comparative morphometric analysis of NMJs was developed and validated (‘NMJ-morph’). NMJ-morph was used to generate baseline data for 2160 NMJs from a single litter of wild type mice, representing 9 distinct muscles across 3 body regions. Principal components analysis (PCA) revealed synaptic size and fragmentation to be the key determinants of synaptic variability. Correlation data revealed the pre-synaptic cell (motor neuron) to be a stronger predictor of synaptic morphology than the post-synaptic cell (muscle fibre). Other factors influencing synaptic variability were in a clear hierarchy: muscle identity accounted for more variation in synaptic form than animal identity, with side having no effect. Human tissue was obtained from 20 patients (aged 34 to 92 years) undergoing lower limb amputation, primarily for the complications of peripheral vascular disease (PVD). Muscle samples were harvested from non-pathological regions of the surgical discard tissue. 2860 human NMJs were analyzed from 4 distinct muscles (extensor digitorum longus, soleus, peroneus longus and peroneus brevis), and compared with equivalent NMJs from wild type mice. Human NMJs displayed unique morphological characteristics, including small size, thin axons, rudimentary nerve terminals and distinctive ‘nummular’ endplates, all of which distinguished them from equivalent mouse NMJs. The previous notion of partial occupancy in human NMJs was disproved. As in mice, the pre-synaptic cell was shown to correlate more strongly with NMJ morphology; in contrast to mice, the human NMJ was found to be relatively stable throughout its 90+ year lifespan. In support of the tissue harvesting procedure, patient co-morbidities (diabetes mellitus and vascular disease) did not significantly impact NMJ morphology. Super-resolution imaging of the NMJ revealed significant differences in the functional architecture of human and mouse active zones. Despite the smaller synaptic size in humans, the total quantity of active zone material was conserved between the species, suggesting a homeostatic mechanism to preserve effective neurotransmission. Parallel proteomic profiling demonstrated further species-specific differences in the broader molecular composition of the NMJ. The cellular and molecular anatomy of the human NMJ is fundamentally different to that of other mammalian species. These differences must be taken into account when translating animal models of disease to the human condition.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:743720 |
| Date | January 2018 |
| Creators | Jones, Ross Alexander |
| Contributors | Gillingwater, Tom ; Kristmundsdottir, Fanney ; Simpson, Hamish |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/29629 |
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