NMJ-Analyser identifies subtle early changes in mouse models of neuromuscular disease

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.

Synapse-specific Lrp4 mRNA enrichment requires Lrp4/MuSK signaling, muscle activity and Wnt non-canonical pathway

Background The neuromuscular junction (NMJ) is a peripheral synapse critical to muscle contraction. Like acetylcholine receptors (AChRs), many essential proteins of NMJ are extremely concentrated at the postjunctional membrane. However, the mechanisms of synapse-specific concentration are not well understood; furthermore, it is unclear whether signaling molecules critical to NMJ formation and maintenance are also locally transcribed. Results We studied the β-gal activity encoded by a lacZ cassette driven by the promoter of the Lrp4 gene. As reported for Lrp4 mRNA, β-gal was in the central region in embryonic muscles and at the NMJ after its formation. However, β-gal was no longer in the central areas of muscle fibers in Lrp4 or MuSK mutant mice, indicating a requirement of Lrp4/MuSK signaling. This phenotype could be rescued by transgenic expression of LRP4 with a transmembrane domain but not soluble ECD in Lrp4 mutant mice. β-gal and AChR clusters were distributed in a broader region in lacZ/ECD than that of heterozygous lacZ/+ mice, indicating an important role of the transmembrane domain in Lrp4 signaling. Synaptic β-gal activity became diffused after denervation or treatment with µ-conotoxin, despite its mRNA was increased, indicating synaptic Lrp4 mRNA enrichment requires muscle activity. β-gal was also diffused in aged mice but became re-concentrated after muscle stimulation. Finally, Lrp4 mRNA was increased in C2C12 myotubes by Wnt ligands in a manner that could be inhibited by RKI-1447, an inhibitor of ROCK in Wnt non-canonical signaling. Injecting RKI-1447 into muscles of adult mice diminished Lrp4 synaptic expression. Conclusions This study demonstrates that synapse-specific enrichment of Lrp4 mRNA requires a coordinated interaction between Lrp4/MuSK signaling, muscle activity, and Wnt non-canonical signaling. Thus, the study provides a new mechanism for Lrp4 mRNA enrichment. It also provides a potential target for the treatment of NMJ aging and other NMJ-related diseases.

Fate of ganglionic synapses and ganglion cell axons during normal and induced cell death.

In order to understand the significance of cell death in the formation of neural circuits, it is necessary to determine whether before cell death neurons have (a) sent axons to the periphery; (b) reached the proper target organs; and (c) have established synaptic connections with them. Axon counts demonstrated that, after sending out initial axons, ciliary cells sprouted numerous collaterals at the time of peripheral synapse formation. Subsequently, large numbers of axons were lost from the nerves, slightly later than the onset of ganglion cell death. A secondary loss of collaterals later occurred unaccompanied by cell death. Measurements of conduction velocity and axon diameters indicated that all ganglion cell axons grew down the proper pathways from the start, but it was not possible to determine whether all axons had actually formed proper synapses. This was ascertained, however, in the ganglion itself where preganglionic fibres were shown to synapse selectively with all ganglion cells before cell death. During this period, degenerating preganglionic synapses were observed on normal cells. It can therefore be inferred that at least some preganglionics established proper synapses before dying and that a single synapse is not sufficient to prevent cell death. In this system neither preganglionic nor ganglionic cell death seems designed to remove improper connections but rather to remove cells that have not competed effectively for a sufficient number of synapses, resulting in a quantitative matching up of neuron numbers.