Identification of novel synaptic components by transcriptome profiling of the murine neuromuscular junction

The neuromuscular junction (NMJ) has been studied for over a century, yet we still do not have a complete picture of all its structural and functional components, knowledge of which is paramount in devising treatment strategies for neuromuscular diseases. Previous microarray-based approaches aimed at elucidating novel NMJ components were hindered by technological limitations. Recent technological advancements propelled next-generation RNA-sequencing with its wider dynamic range to the forefront of transcriptome-level gene expression profiling.
We utilized laser-capture microdissection to isolate myonuclei underlying the NMJ combined with RNA-sequencing and successfully generated NMJ gene expression profiles of fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles and identified a large number of potential novel NMJ genes. The expression levels of canonical NMJ genes were nearly identical between the EDL and SOL, which suggests that the core NMJ gene program might be well conserved between different skeletal muscle types. We used in vivo muscle electroporation to overexpress one of our candidate genes, the transcription factor T-box 21 (TBX21), in the tibialis anterior (TA) muscle and observed an increased density of postsynaptic acetylcholine receptors. TBX21 may thus represent a novel transcription factor contributing to the regulation of the NMJ gene program, with a role in postsynaptic sensitivity.
We also generated NMJ gene expression profiles of the TA muscle of 10-month-old (“young”) and 30-month-old (“old”) mice to investigate the effect of aging on the NMJ gene program. Strikingly, the NMJ gene program was remarkably stable, with nearly identical expression levels of canonical NMJ genes between young and old mice. This implies that age-related perturbations of the NMJ are likely caused by external factors, such as accumulated myofiber damage and changes in nerve input, rather than by gradual dysregulation of the NMJ gene program with increasing age. Our findings argue against the hypothesis that aging leads to a broad deterioration of the NMJ gene program that would contribute to perturbations of NMJ structure and function.
Furthermore, functional annotation analysis of our different NMJ gene expression datasets strongly indicates the importance of an extensive number of hitherto unknown glycoproteins, as well as of posttranslational modifications, especially glycosylations, at the synaptic basal lamina. We highlight a set of candidate genes that encode for enzymes putatively involved in these processes at the NMJ, and which are potentially involved in the pathophysiology of neuromuscular diseases such as congenital myasthenic syndromes. This thesis expands our understanding of the complexity of the NMJ and lays the foundation for further research that will functionally characterize novel synaptic components and provide the basis for novel therapeutic treatment strategies.