Investigating the impact of pathological conditions and gut microbiota on the structure and function of the neuromuscular system

The neuromuscular system functionally links the nervous system to skeletal muscle through chemical signalling at the neuromuscular junction (NMJ). The coordinated activity of all these components is vital for the accomplishment of voluntary and non-voluntary movements, while their dysfunction results in severe neuromuscular disorders. Collagen VI (ColVI)-related myopathies are rare inherited muscular dystrophies caused by mutations in the extracellular matrix protein ColVI, for which no cure currently exists. A novel perspective for their treatment recently emerged with the acknowledgement of ColVI role in the structural and functional maintenance of the NMJ. Hence, during the first part of my PhD, I investigated the NMJ as a novel potential druggable target for ColVI-related myopathies. To address this, I examined the effects of salbutamol, an FDA-approved b2-adrenergic agonist safely and successfully repurposed in numerous NMJ-related disorders, on the structural and electrophysiological NMJ defects and on the myopathic phenotype of a preclinical model of these disorders, the ColVI knockout mouse (Col6a1-/-). Our results showed that one-month systemic salbutamol administration led to an expansion of the postsynaptic endplate as well as to a recovery of NMJ fragmentation and altered neurotransmission. Salbutamol also mitigated muscle wasting by inducing hypertrophy and ameliorating muscle force at physiologically relevant stimulation frequencies. Although a refinement of the adopted therapeutic regimen is warranted to avoid undesired receptor desensitization, our data point at salbutamol as a promising candidate for counteracting ColVI-related myopathies disease progression. As a second part of my PhD, I contributed to explore the potential interaction between the gut microbiota (GM) and all the components of the neuromuscular system. Indeed, the GM has recently been recognized as a positive modulator of skeletal muscle homeostasis. However, GM impact on skeletal muscles during the neonatal stage, as well as its influence on their functional dependence on innervating somatic nerves and on the somatic peripheral nervous system (PNS) itself has not been investigated. To fill this gap, we analyzed peripheral nerves, skeletal muscles and NMJs from neonatal and young adult mouse models with various GM composition, such as germ free (GF), gnotobiotic mice (colonized with 12 known bacterial strains, OMM12), conventional mice (with a complex GM, CGM) and ex-GF mice (recolonized, after weaning, with a complex GM). Our data showed that absence of a GM impairs proper development of peripheral nerves and their motor targets. Indeed, young adult GF mice displayed atrophic skeletal muscles, deregulation of genes involved in muscle mass regulation, increased levels of basal autophagy and imbalanced protein turnover. Smaller muscle fibers were already detected at the neonatal stage due to a slower muscle development. Consistently, neonatal GF muscles exhibited less developed NMJs and downregulation of pathways related to synaptic activity. At the adult stage, the GF status resulted in NMJs with a fragmented endplate and an expanded presynaptic compartment. A differential expression of genes involved in PNS development and myelination was also detected in GF neonatal and young adult peripheral nerves, which, at the adult stage, were also characterised by the presence of smaller and hypermyelinated axons. Reconstitution of GF mice with the simplified OMM12 bacterial community allowed only a partial correction of the aberrant PNS-skeletal muscle development. Of note, ex-GF mice, while able to almost completely rescue muscular and NMJ postsynaptic alterations, failed to correct the aberrancies of the NMJ pre-synaptic compartment and of somatic nerves. Overall, our data acknowledge a novel axis, mediating GM impact on the neuromuscular system. Which GM-derived factors are directly mediating this interaction is under current investigation.