Abstract
The neuromuscular junction (NMJ) is the peripheral synapse formed between a motor neuron axon terminal and a muscle fibre. NMJs are thought to be the primary site of peripheral pathology in many neuromuscular diseases, but innervation/denervation status is often assessed qualitatively with poor systematic criteria across studies, and separately from 3D morphological structure. Here, we describe the development of ‘NMJ-Analyser’, to comprehensively screen the morphology of NMJs and their corresponding innervation status automatically. NMJ-Analyser generates 29 biologically relevant features to quantitatively define healthy and aberrant neuromuscular synapses and applies machine learning to diagnose NMJ degeneration. We validated this framework in longitudinal analyses of wildtype mice, as well as in four different neuromuscular disease models: three for amyotrophic lateral sclerosis (ALS) and one for peripheral neuropathy. We showed that structural changes at the NMJ initially occur in the nerve terminal of mutant TDP43 and FUS ALS models. Using a machine learning algorithm, healthy and aberrant neuromuscular synapses are identified with 95% accuracy, with 88% sensitivity and 97% specificity. Our results validate NMJ-Analyser as a robust platform for systematic and structural screening of NMJs, and pave the way for transferrable, and cross-comparison and high-throughput studies in neuromuscular diseases.
Highlights
Protein expressionAge Numbers/sex Disease state RefSOD1G93A/+ MGI: 2448770 Transgenic C57BL/6N-SJL1 ~ 20-fold overexpression 1.5GarsC201R/+ MGI: 3760297 ENU mutagenesis C57BL/6J
We focused on the 12-month old FUSΔ14/+ and TDP43M323K/M323K strains because, these mice did not have significant changes in neuromuscular junction (NMJ) innervation status, we observed significant alteration of ‘non-compactness’ and ‘volume/surface ratio’ in the pre-synaptic component of both strains, but not in motor endplates (Fig. 3b,c)
Comparative studies of innervation status and structural analysis are limited, which diminishes our understanding of NMJs in health, ageing and pathology
Summary
Cluster_dist Cluster_size Cluster_numbers Fragmentation Non-compactness Shape factor Rugosity, internal Rugosity, external Length (μm) Surface/volume ratio. Morphological parameters measuring the interaction between nerve terminal and endplate showed that midand late-mature NMJ structures were conserved and vary significantly from early-mature architecture (Fig. 2c). These results were similar in the C57BL/6-SJL mice (Additional File 1: Fig. S3). (b) Matrix correlation plot of nerve terminal and motor endplate parameters, and interaction between them measured in early-, mid- and late-mature NMJs. Positive and negative correlations are given by blue and red scales, respectively (P value ≤ 5 × 1 0−2). We explored whether NMJ-Analyser, NMJ-Morph and Volocity could detect mild structural variations in neuromuscular synapses by comparing nerve terminal and endplate outputs (Fig. 4d,e).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
