Phd in MORPHOGENESIS AND TISSUE ENGINEERING

Thesis title: Development of innovative techniques, experimental devices and testing protocols for the measurement of muscle and neuromuscular junction functionality in Amyotrophic Lateral Sclerosis

Amyotrophic Lateral Sclerosis (ALS) remains an invariably fatal disease, in which neuromuscular junction (NMJ) functionality is strongly impaired. To this, the aim of this research project was to develop a series of novel testing tools for a precise assessment of the altered communication between muscle and nerve in ALS progression. A novel technique for the in-situ measurement of murine Tibialis Anterior (TA) NMJ functionality in isotonic conditions was developed and validated. A novel parameter, named Isotonic Neurotransmission Failure (INF), was proposed. Results showed an increase in INF of SOD1G93A mouse TA muscles at the end-stage of the disease, highlighting, for the first time, an increased impairment of NMJ functionality in isotonic conditions. An embedded system for the measurement of 3D engineered skeletal muscle tissues’ contractility with a non-invasive technique was proposed. Results showed the capability of the system to not impair tissue's contractility during the entire growth, and to discriminate healthy and pathological conditions. Finally, a 3D microfluidic device was designed and realized to promote the formation of NMJ between spinal cord-derived neuronal cells and 3D engineered skeletal muscle. Results showed a good attraction between these two cells populations, paving the basis for the development of a more comprehensive 3D NMJ in-vitro model. On the other hand, since extracellular vesicles (EVs) are involved in ALS pathological proteins transportation, a series of preliminary experiments with muscle cells’ populations was carried out, with the final aim of evaluating the role of SOD1G93A mice-derived EVs on the novel experimental models here proposed. Results showed that SOD1G93A mice-derived EVs increased in number during the ALS progression, and impaired C2C12 cells’ differentiation. In conclusion, a series of novel testing tools have been developed for a precise assessment of the NMJ functionality in different models which, of note, can be also employed to unravel the mechanism behind muscle-nerve impairments in other neurodegenerative pathologies.